Fadd-like anti-apoptotic molecules, flame-1 and flame-2, methods of using the same, and compositions for and methods of making the same

ABSTRACT

Two FADD-like anti-apoptotic proteins FLAME-1 and FLAME-2 that regulate Fas/TNFR1- or UV-induced apoptosis are disclosed. Nucleotide sequences encoding the proteins are disclosed as are methods of using the nucleic acid molecules and making the proteins. Pharmaceutical compositions and methods of using the same are disclosed. Reagents, kits and methods of identifying compounds that inhibit anti-apoptotic activity of the proteins and methods of identifying compounds that inhibit binding activity of the proteins are disclosed.

ACKNOWLEDGMENT OF GOVERNMENT RIGHTS

This invention was made with Government support under Grant AG 13487from the National Institutes of Health. The Government has certainrights in this invention.

This Application is a divisional of U.S. application Ser. No. 08/859,167filed May 20,1997 entitled FADD-LIKE ANTI-APOPTOTIC MOLECULES, METHODSOF USING THE SAME, AND COMPOSITIONS FOR AND METHODS Of MAKING THE SAME.

FIELD OF THE INVENTION

The invention relates to the identification and cloning of two FADD-likeanti-apoptotic molecules that regulate Fas/TNFR1- or UV-inducedapoptosis, to methods of using the same, and to compositions for andmethods of making the same and to methods of making and using the same.

BACKGROUND OF THE INVENTION

Apoptotic cell death is essential for normal development and maintenanceof normal tissue size homeostasis in multicellular organisms. There isgrowing evidence that dysregulation of apoptosis may lead to severalhuman diseases including cancer and degenerative neuronal diseases suchas Alzheimer's and Parkinson's diseases.

Several members of the caspase family of proteases (Alnemri, E. S. etal. 1996 Cell 87, 171, which is incorporated herein by reference) havebeen implicated as key regulators of programmed cell death or apoptosis(Alnemri, E. S. 1997 J. Cell. Biochem. 64, 33-42 and Henkart, P. A. 1996Immunity 4, 195-201 which are incorporated herein be reference). Thepro-apoptotic caspases can be divided into two groups: those with alarge prodomain such as ICH-1 (caspase-2), Mch4 (caspase-10),Mch5/MACH/FLICE (caspase-8) and Mch6/ICE-Lap-6 (caspase-9) and thosewith a small prodomain such as CPP32/YAMA/Apopain (caspase-3), Mch2(caspase-6) and Mch3/ICE-Lap-3 (caspase-7). Caspases with largeprodomains are probably the most upstream caspases. They are recruitedby several death-signaling receptors that belong to the TNFR family,through interactions of their prodomain with the receptor-interactingadaptor molecules FADD/Mort1 or CRADD/RAIDD. For example, the prodomainsof Mch4 and Mch5 contain two tandem regions that show significanthomology with the N-terminal death effector domain (DED) of FADD.Engagement of Fas/TNFR1 results in recruitment of FADD to the receptorcomplex, which presumably triggers activation of the caspase apoptoticpathway through interaction of its DED with the corresponding motifs inthe prodomain of Mch5 and probably Mch4. CRADD presumably functions likeFADD by recruiting ICH-1 to the Fas/TNFR1 complex, through interactionof its N-terminal domain with the corresponding motif in the prodomainof ICH-1. Thus, the prodomains of caspases function to physically linkthe death receptors to the downstream caspase activation pathway.

There is a need to identify proteins that regulate apoptosis. There is aneed for isolated FADD-like anti-apoptotic molecules that regulateFas/TNFR1- or UV-induced apoptosis, and for compositions and methods ofproducing and isolating FADD-like anti-apoptotic molecules that regulateFas/TNFR1- or UV-induced apoptosis. There is a need to isolated proteinsthat are FADD-like anti-apoptotic molecules that regulate Fas/TNFR1- orUV-induced apoptosis. There is a need to isolated nucleic acid moleculesthat encode FADD-like anti-apoptotic molecules that regulate Fas/TNFR1-or UV-induced apoptosis. There is a need for compounds which inhibitactivity of FADD-like anti-apoptotic molecules that regulate Fas/TNFR1-or UV-induced apoptosis. There is a need for kits and methods ofidentifying such compounds.

SUMMARY OF THE INVENTION

The invention relates to substantially pure proteins that have aminoacid sequences shown in SEQ ID NO:2 or SEQ ID NO:4.

The invention relates to pharmaceutical compositions comprising aprotein that has the amino acid sequence shown in SEQ ID NO:2 or SEQ IDNO:4 in combination with a pharmaceutically acceptable carrier.

The invention relates to isolated nucleic acid molecules that comprisenucleic acid sequences that encode a protein that has an amino acidsequence shown in SEQ ID NO:2 or SEQ ID NO:4.

The invention relates to pharmaceutical compositions that comprisenucleic acid molecule that comprise nucleic acid sequences that encode aprotein that has an amino acid sequence shown in SEQ ID NO:2 or SEQ IDNO:4 in combination with a pharmaceutically acceptable carrier.

The invention relates to isolated nucleic acid molecules that consist ofSEQ ID NO:1 or SEQ ID NO:3 or a fragment thereof having at least 5nucleotides.

The invention relates to a recombinant expression vector comprising thenucleic acid molecule that has a nucleotide sequence that comprises SEQID NO:1 or SEQ ID NO:3.

The invention relates to a host cell comprising a recombinant expressionvector comprising the nucleic acid molecule that has a nucleotidesequence that comprises SEQ ID NO:1 or SEQ ID NO:3.

The invention relates to an oligonucleotide molecule comprising anucleotide sequence complimentary to a nucleotide sequence of at least 5nucleotides of SEQ ID NO:1 or SEQ ID NO:3.

The invention relates to isolated antibodies that bind to an epitope onSEQ ID NO:2 and/or SEQ ID NO:4.

The invention relates to methods of identifying substrates, activatorsor inhibitors of FLAME-1 and/or FLAME-2.

The invention relates to methods of inhibiting expression of FLAME-1and/or FLAME-2 by contacting cells that express FLAME-1 and/or FLAME-2with a nucleic acid molecule that comprises an antisense nucleotidesequence that prevents transcription of FLAME-1 and/or FLAME-2 genesequences or translation of FLAME-1 and/or FLAME-2 mRNA.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E disclose sequence analysis, tissue distribution andchromosomal localization data of FLAMEs. FIG. 1A shows the predictedamino acid sequence of FLAME-1 compared to Mch5-beta and Mch4 andFLAME-I's structure. FIG. 1B shows the predicted amino acid sequence ofFLAME-2 and its structure. FIG. 1C shows the N-terminal region ofFLAME-2 (amino acids 23-101) shares significant homology with the FDH-Aof Mch5b and the N-terminal DED of FADD. FIG. 1D shows results fromNorthern blot analysis of FLAME-1 and FLAME-2 mRNAs. FIG. 1E showsFLAME-1, Mch4 and Mch5 genes are localized to chromosome 2q33-34.

FIGS. 2A-2E shows in vitro interactions of FLAME-1 and FLAME-2. FIG. 2Ashows cleavage of FLAME-1 by caspases. FIGS. 2B-2E show in vitrointeractions.

FIGS. 3A-3F show in vivo interactions of FLAME-1 and FLAME-2.

FIGS. 4A and 4B show FLAME-1 and FLAME-2 protect cells againstapoptosis.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "FLAMEs" is meant to refer to the two FADD-likeapoptotic/anti-apoptotic molecules that have been isolated and clonedand discovered to regulate Fas/TNFR1- or UV-induced apoptosis.

As used herein, FLAME-1 refers to one of the two FLAMEs. The amino acidsequence of FLAME-1 is set forth in SEQ ID NO:2. The cloned cDNA whichencodes FLAME-1 is set forth in SEQ ID NO:1.

As used herein, FLAME-2 refers to one of the two FLAMEs. The amino acidsequence of FLAME-2 is set forth in SEQ ID NO:4. The cloned cDNA whichencodes FLAME-2 is set forth in SEQ ID NO:3.

Two novel human anti-apoptotic proteins that contain FADD/Mort1DED-homology regions, designated FLAME-1 and FLAME-2 have beenidentified and cloned. FLAME-1, although most similar in structure toMch4 and Mch5, does not possess caspase activity, but can interactspecifically with FADD, Mch4, Mch5 and FLAME-2. FLAME-1 is recruited tothe Fas receptor complex and can abrogate Fas/TNF-induced apoptosis uponexpression in Fas/TNF-sensitive MCF-7 cells. FLAME-2, on the other hand,is similar in structure to FADD, but its C-terminal region does not havea death domain homology. It interacts weakly with Mch4 and Mch5 but doesnot interact with FADD. It can abrogate UV-induced apoptosis and to alesser degree inhibit Fas/TNFR-induced apoptosis in the same cell line.These findings identify two novel endogenous control points thatregulate Fas/TNFR1- and UV-mediated apoptosis.

The discovery of the two FLAMEs provides the means to design anddiscover specific inhibitors, activators and substrates of theseanti-apoptotic molecules. According to the present invention, FLAMEs maybe used to screen compounds for inhibitors, activators or substrates.Inhibitors are useful as apoptotic agents. Activators are useful asanti-apoptotic agents. FLAME-1 and FLAME-2 proteins are useful asreagents in assays to identify inhibitors and activators as well as inbinding assays such as FLAME-1 binding assays with FADD, Mch4, Mch5 andFLAME-2 and FLAME-2 binding assays with Mch4, Mch5 and FLAME-1. FLAME-1may also be useful as a substrate for caspase in assays to identifycaspase inhibitors. Kits are provided for screening compounds for FLAMEsinhibitors. Kits are provided for screening compounds for FLAMEsactivators. Kits are provided for screening compounds for FLAME bindingassays. The nucleotide sequences that encode the FLAMEs are disclosedherein and allow for the production of pure protein, the design ofprobes which specifically hybridize to nucleic acid molecules thatencode the FLAMEs and antisense compounds to inhibit transcription ofFLAMEs. Anti-FLAME-1 and anti-FLAME-2 antibodies are provided.Anti-FLAME-1 antibodies may be inhibitors of FLAME-1 and may be used inmethods of isolating pure FLAME-1 and methods of inhibiting FLAME-1activity. Anti-FLAME-2 antibodies may be inhibitors of FLAME-2 and maybe used in methods of isolating pure FLAME-2 and methods of inhibitingFLAME-1 activity.

The present invention provides substantially purified FLAMEs, FLAME-1and FLAME-2 which have amino acid sequences consisting of: SEQ ID NO:2and SEQ ID NO:4, respectively. FLAME-1 and FLAME-2 can be isolated fromnatural sources, produced by recombinant DNA methods or synthesized bystandard protein synthesis techniques.

Antibodies which specifically bind to a particular FLAME may be used topurify the protein from natural sources using well known techniques andreadily available starting materials. Such antibodies may also be usedto purify the FLAME from material present when producing the protein byrecombinant DNA methodology. The present invention relates to antibodiesthat bind to an epitope which is present on a FLAME selected from thegroup consisting of: FLAME-1--SEQ ID NO:2 and FLAME-2--SEQ ID NO:4. Asused herein, the term "antibody" is meant to refer to complete, intactantibodies, and Fab fragments and F(ab)₂ fragments thereof. Complete,intact antibodies include monoclonal antibodies such as murinemonoclonal antibodies, chimeric antibodies and humanized antibodies. Insome embodiments, the antibodies specifically bind to an epitope of onlyone of: FLAME-1 and FLAME-2. Antibodies that bind to an epitope which ispresent on a FLAME are useful to isolate and purify the FLAME from bothnatural sources or recombinant expression systems using well knowntechniques such as affinity chromatography. Such antibodies are usefulto detect the presence of such protein in a sample and to determine ifcells are expressing the protein.

The production of antibodies and the protein structures of complete,intact antibodies, Fab fragments and F(ab)₂ fragments and theorganization of the genetic sequences that encode such molecules arewell known and are described, for example, in Harlow, E. and D. Lane(1988) ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. which is incorporated herein by reference.Briefly, for example, the FLAME-1 or FLAME-2 protein, or an immunogenicfragment thereof is injected into mice. The spleen of the mouse isremoved, the spleen cells are isolated and fused with immortalized mousecells. The hybrid cells, or hybridomas, are cultured and those cellswhich secrete antibodies are selected. The antibodies are analyzed and,if found to specifically bind to the FLAME, the hybridoma which producesthem is cultured to produce a continuous supply of antibodies.

Using standard techniques and readily available starting materials, anucleic acid molecule that encodes each of the FLAMEs may be isolatedfrom a cDNA library, using probes or primers which are designed usingthe nucleotide sequence information disclosed in SEQ ID NO:1 or SEQ IDNO:3. The present invention relates to an isolated nucleic acid moleculethat comprises a nucleotide sequence that encodes a FLAME selected fromthe group consisting of FLAME-1 and FLAME-2 that comprises the aminoacid sequence of SEQ ID NO:2 and SEQ ID NO:4, respectively. In someembodiments, the nucleic acid molecules consist of a nucleotide sequencethat encodes FLAME-1 or FLAME-2. In some embodiments, the nucleic acidmolecules comprise the nucleotide sequence that consists of the codingsequence in SEQ ID NO:1 or SEQ ID NO:3. In some embodiments, the nucleicacid molecules consist of the nucleotide sequence set forth in SEQ IDNO:1 or SEQ ID NO:3. The isolated nucleic acid molecules of theinvention are useful to prepare constructs and recombinant expressionsystems for preparing the FLAMEs of the invention.

A cDNA library may be generated by well known techniques. A cDNA clonewhich contains one of the nucleotide sequences set out is identifiedusing probes that comprise at least a portion of the nucleotide sequencedisclosed in SEQ ID NO:1 or SEQ ID NO:3. The probes have at least 16nucleotides, preferably 24 nucleotides. The probes are used to screenthe cDNA library using standard hybridization techniques. Alternatively,genomic clones may be isolated using genomic DNA from any human cell asa starting material. In either cDNA or genomic probes, the sequence ofthe probe is unique to the FLAME that it is designed to hybridize to.That is, the sequence is selected to be unique relative to other knownsequences. Unique sequences may be identified by comparing the sequencesset forth in SEQ ID NO:1 and SEQ ID NO:3 to each other and to thesequences set forth in sequence data bases such as Genbank. Uniquefragments of SEQ ID NO:1 and SEQ ID NO:3 are useful because they canhybridize to clones without cross hybridizing to other non-FLAMEencoding sequences.

The present invention relates to isolated nucleic acid molecules thatcomprise a nucleotide sequence identical or complementary to a uniquefragment of SEQ ID NO:1 or SEQ ID NO:3 which is at least 10 nucleotides.In some embodiments, the isolated nucleic acid molecules consist of aunique nucleotide sequence identical or complementary to a fragment ofSEQ ID NO:1 or SEQ ID NO:3 which is at least 10 nucleotides. In someembodiments, the isolated nucleic acid molecules comprise or consist ofnucleotide sequence identical or complementary to a unique fragment ofSEQ ID NO:1 or SEQ ID NO:2 which is 15-150 nucleotides. In someembodiments, the isolated nucleic acid molecules comprise or consist ofa nucleotide sequence identical or complementary to a unique fragment ofSEQ ID NO:1 or SEQ ID NO:3 which is 15-30 nucleotides. Isolated nucleicacid molecules that comprise or consist of a nucleotide sequenceidentical or complementary to a fragment of SEQ ID NO:1 or SEQ ID NO:3which is at least 10 nucleotides are useful-as probes for identifyinggenes and cDNA sequence having SEQ ID NO:1 or SEQ ID NO:3, respectively,PCR primers for amplifying genes and cDNA having SEQ ID NO:1 or SEQ IDNO:3, respectively, and antisense molecules for inhibiting transcriptionand translation of genes and cDNA, respectively, which encode FLAMEshaving the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4,respectively.

The cDNA that encodes FLAME-1 or FLAME-2 may be used as a molecularmarker in electrophoresis assays in which cDNA from a sample isseparated on an electrophoresis gel and FLAME probes are used toidentify bands which hybridize to such probes. Specifically, SEQ ID NO:1or portions thereof, or SEQ ID NO:3 or portions thereof, may be used asa molecular marker in electrophoresis assays in which cDNA from a sampleis separated on an electrophoresis gel and FLAME-specific probes areused to identify bands which hybridize to them, indicating that the bandhas a nucleotide sequence complementary to the sequence of the probes.The isolated nucleic acid molecule provided as a size marker will showup as a positive band which is known to hybridize to the probes and thuscan be used as a reference point to the size of cDNA that encodesFLAME-1 and FLAME-2, respectively. Electrophoresis gels useful in suchan assay include standard polyacrylamide gels as described in Sambrooket al., Molecular Cloning a Laboratory Manual, Second Ed. Cold SpringHarbor Press (1989) which is incorporated herein by reference.

The nucleotide sequences in SEQ ID NO:1 and SEQ ID NO:3 may be used todesign probes, primers and complimentary molecules which specificallyhybridize to the unique nucleotide sequences of FLAME-1 and FLAME-2,respectively. Probes, primers and complimentary molecules whichspecifically hybridize to nucleotide sequence that encodes FLAME-1 andFLAME-2 may be designed routinely by those having ordinary skill in theart.

The present invention also includes labeled oligonucleotides which areuseful as probes for performing oligonucleotide hybridization methods toidentify FLAME-1 and FLAME-2. Accordingly, the present inventionincludes probes that can be labeled and hybridized to unique nucleotidesequences of FLAME-1 and FLAME-2. The labeled probes of the presentinvention are labeled with radiolabelled nucleotides or are otherwisedetectable by readily available nonradioactive detection systems. Insome preferred embodiments, probes comprise oligonucleotides consistingof between 10 and 100 nucleotides. In some preferred, probes compriseoligonucleotides consisting of between 10 and 50 nucleotides. In somepreferred, probes comprise oligonucleotides consisting of between 12 and20 nucleotides. The probes preferably contain nucleotide sequencecompletely identical or complementary to a fragment of a uniquenucleotide sequences of FLAME-1 and FLAME-2.

PCR technology is practiced routinely by those having ordinary skill inthe art and its uses in diagnostics are well known and accepted. Methodsfor practicing PCR technology are disclosed in "PCR Protocols: A Guideto Methods and Applications", Innis, M. A., et al. Eds. Academic Press,Inc. San Diego, Calif. (1990) which is incorporated herein by reference.Applications of PCR technology are disclosed in "Polymerase ChainReaction" Erlich, H. A., et al., Eds. Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989) which is incorporated herein by reference.Some simple rules aid in the design of efficient primers. Typicalprimers are 18-28 nucleotides in length having 50% to 60% g+ccomposition. The entire primer is preferably complementary to thesequence it must hybridize to. Preferably, primers generate PCR products100 basepairs to 2000 base pairs. However, it is possible to generateproducts of 50 base pairs to up to 10 kb and more.

PCR technology allows for the rapid generation of multiple copies ofnucleotide sequences by providing 5' and 3' primers that hybridize tosequences present in a nucleic acid molecule, and further providing freenucleotides and an enzyme which fills in the complementary bases to thenucleotide sequence between the primers with the free nucleotides toproduce a complementary strand of DNA. The enzyme will fill in thecomplementary sequences adjacent to the primers. If both the 5' primerand 3' primer hybridize to nucleotide sequences on the complementarystrands of the same fragment of nucleic acid, exponential amplificationof a specific double-stranded product results. If only a single primerhybridizes to the nucleic acid molecule, linear amplification producessingle-stranded products of variable length.

One having ordinary skill in the art can isolate the nucleic acidmolecule that encode FLAME-1 or FLAME-2 and insert it into an expressionvector using standard techniques and readily available startingmaterials.

The present invention relates to a recombinant expression vector thatcomprises a nucleotide sequence that encodes FLAME-1 or FLAME-2 thatcomprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4,respectively. As used herein, the term "recombinant expression vector"is meant to refer to a plasmid, phage, viral particle or other vectorwhich, when introduced into an appropriate host, contains the necessarygenetic elements to direct expression of the coding sequence thatencodes the FLAMEs of the invention. The coding sequence is operablylinked to the necessary regulatory sequences. Expression vectors arewell known and readily available. Examples of expression vectors includeplasmids, phages, viral vectors and other nucleic acid molecules ornucleic acid molecule containing vehicles useful to transform host cellsand facilitate expression of coding sequences. In some embodiments, therecombinant expression vector comprises the nucleotide sequence setforth in SEQ ID NO:1 or SEQ ID NO:3. The recombinant expression vectorsof the invention are useful for transforming hosts to preparerecombinant expression systems for preparing the FLAMEs of theinvention.

The present invention relates to a host cell that comprises therecombinant expression vector that includes a nucleotide sequence thatencodes a FLAME that comprises SEQ ID NO:1 or SEQ ID NO:3. In someembodiments, the host cell comprises a recombinant expression vectorthat comprises SEQ ID NO:1 or SEQ ID NO-:3. Host cells for use in wellknown recombinant expression systems for production of proteins are wellknown and readily available. Examples of host cells include bacteriacells such as E. coli, yeast cells such as S. cerevisiae, insect cellssuch as S. frugiperda, non-human mammalian tissue culture cells chinesehamster ovary (CHO) cells and human tissue culture cells such as HeLacells.

The present invention relates to a transgenic non-human mammal thatcomprises the recombinant expression vector that comprises a nucleicacid sequence that encodes a FLAME that comprises the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:4. Transgenic non-human mammalsuseful to produce recombinant proteins are well known as are theexpression vectors necessary and the techniques for generatingtransgenic animals. Generally, the transgenic animal comprises arecombinant expression vector in which the nucleotide sequence thatencodes a FLAME of the invention is operably linked to a mammary cellspecific promoter whereby the coding sequence is only expressed inmammary cells and the recombinant protein so expressed is recovered fromthe animal's milk. In some embodiments, the coding sequence that encodesa FLAME is SEQ ID NO:1 or SEQ ID NO:3.

In some embodiments, for example, one having ordinary skill in the artcan, using well known techniques, insert such DNA molecules into acommercially available expression vector for use in well knownexpression systems. For example, the commercially available plasmidpSE420 (Invitrogen, San Diego, Calif.) may be used for production ofcollagen in E. coli. The commercially available plasmid pYES2(Invitrogen, San Diego, Calif.) may, for example, be used for productionin S. cerevisiae strains of yeast. The commercially available MAXBAC™complete baculovirus expression system (Invitrogen, San Diego, Calif.)may, for example, be used for production in insect cells. Thecommercially available plasmid pcDNA I (Invitrogen, San Diego, Calif.)may, for example, be used for production in mammalian cells such asChinese Hamster Ovary cells. One having ordinary skill in the art canuse these commercial expression vectors and systems or others to produceFLAME of the invention using routine techniques and readily availablestarting materials. (See e.g., Sambrook et al., Molecular Cloning aLaboratory Manual, Second Ed. Cold Spring Harbor Press (1989) which isincorporated herein by reference.) Thus, the desired proteins can beprepared in both prokaryotic and eukaryotic systems, resulting in aspectrum of processed forms of the protein.

One having ordinary skill in the art may use other commerciallyavailable expression vectors and systems or produce vectors using wellknown methods and readily available starting materials. Expressionsystems containing the requisite control sequences, such as promotersand polyadenylation signals, and preferably enhancers, are readilyavailable and known in the art for a variety of hosts. See e.g.,Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. ColdSpring Harbor Press (1989).

A wide variety of eukaryotic hosts are also now available for productionof recombinant foreign proteins. As in bacteria, eukaryotic hosts may betransformed with expression systems which produce the desired proteindirectly, but more commonly signal sequences are provided to effect thesecretion of the protein. Eukaryotic systems have the additionaladvantage that they are able to process introns which may occur in thegenomic sequences encoding proteins of higher organisms. Eukaryoticsystems also provide a variety of processing mechanisms which result in,for example, glycosylation, carboxy-terminal amidation, oxidation orderivatization of certain amino acid residues, conformational control,and so forth.

Commonly used eukaryotic systems include, but is not limited to, yeast,fungal cells, insect cells, mammalian cells, avian cells, and cells ofhigher plants. Suitable promoters are available which are compatible andoperable for use in each of these host types as well as are terminationsequences and enhancers, e.g. the baculovirus polyhedron promoter. Asabove, promoters can be either constitutive or inducible. For example,in mammalian systems, the mouse metallothionein promoter can be inducedby the addition of heavy metal ions.

The particulars for the construction of expression systems suitable fordesired hosts are known to those in the art. Briefly, for recombinantproduction of the protein, the DNA encoding the polypeptide is suitablyligated into the expression vector of choice. The DNA is operably linkedto all regulatory elements which are necessary for expression of the DNAin the selected host. One having ordinary skill in the art can, usingwell known techniques, prepare expression vectors for recombinantproduction of the polypeptide.

The expression vector including the DNA that encodes a FLAME is used totransform the compatible host which is then cultured and maintainedunder conditions wherein expression of the foreign DNA takes place. Theprotein of the present invention thus produced is recovered from theculture, either by lysing the cells or from the culture medium asappropriate and known to those in the art. One having ordinary skill inthe art can, using well known techniques, isolate the FLAME that isproduced using such expression systems. The methods of purifying FLAMEsfrom natural sources using antibodies which specifically bind to theFLAME as described above, may be equally applied to purifying FLAMEsproduced by recombinant DNA methodology.

Examples of genetic constructs include a FLAME coding sequence operablylinked to a promoter that is functional in the cell line into which theconstructs are transfected. Examples of constitutive promoters includepromoters from cytomegalovirus or SV40. Examples of inducible promotersinclude mouse mammary leukemia virus or metallothionein promoters. Thosehaving ordinary skill in the art can readily produce genetic constructsuseful for transfecting with cells with DNA that encodes a FLAME fromreadily available starting materials. Such gene constructs are usefulfor the production of the FLAME.

In some embodiments of the invention, transgenic non-human animals aregenerated. The transgenic animals according to the invention contain SEQID NO:1 or SEQ ID NO:3 under the regulatory control of a mammaryspecific promoter. One having ordinary skill in the art using standardtechniques, such as those taught in U.S. Pat. No. 4,873,191 issued Oct.10, 1989 to Wagner and U.S. Pat. No. 4,736,866 issued Apr. 12, 1988 toLeder, both of which are incorporated herein by reference, can producetransgenic animals which produce the Mch2 isoform. Preferred animals arerodents, particularly goats, rats and mice.

In addition to producing these proteins by recombinant techniques,automated peptide synthesizers may also be employed to produce FLAMEs ofthe invention. Such techniques are well known to those having ordinaryskill in the art and are useful if derivatives which have substitutionsnot provided for in DNA-encoded protein production.

FLAMEs may be used as a pharmaceutical to inhibit apoptosis. Similarly,nucleic acid molecules that encode FLAMEs may be used as part ofpharmaceutical compositions for gene therapy. Diseases characterized byapoptosis include HIV infection and Alzheimer's disease. Those havingordinary skill in the art can readily identify individuals who aresuspected of suffering from such diseases, conditions and disordersusing standard diagnostic techniques.

Pharmaceutical compositions according to the invention comprise apharmaceutically acceptable carrier in combination with FLAME-1 orFLAME-2, or a nucleic acid molecule that encodes FLAME-1 or FLAME-2.Pharmaceutical formulations are well known and pharmaceuticalcompositions comprising FLAME-1 or FLAME-2, or a nucleic acid moleculethat encodes FLAME-1 or FLAME-2 may be routinely formulated by onehaving ordinary skill in the art. Suitable pharmaceutical carriers aredescribed in Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field, which is incorporated herein by reference.The present invention relates to an injectable pharmaceuticalcomposition that comprises a pharmaceutically acceptable carrier andFLAME-1 or FLAME-2, or a nucleic acid molecule that encodes FLAME-1 orFLAME-2. Some embodiments of the invention relate to injectablepharmaceutical compositions that comprise a pharmaceutically acceptablecarrier and amino acid sequence SEQ ID NO:2 or SEQ ID NO:4. FLAME-1 orFLAME-2 is preferably sterile and combined with a sterile pharmaceuticalcarrier.

In some embodiments, for example, FLAME-1 or FLAME-2 can be formulatedas a solution, suspension, emulsion or lyophilized powder in associationwith a pharmaceutically acceptable vehicle. Examples of such vehiclesare water, saline, Ringer's solution, dextrose solution, and 5% humanserum albumin. Liposomes and nonaqueous vehicles such as fixed oils mayalso be used. The vehicle or lyophilized powder may contain additivesthat maintain isotonicity (e.g., sodium chloride, mannitol) and chemicalstability (e.g., buffers and preservatives). The formulation issterilized by commonly used techniques.

An injectable composition may comprise FLAME-1 or FLAME-2 in a dilutingagent such as, for example, sterile water, electrolytes/dextrose, fattyoils of vegetable origin, fatty esters, or polyols, such as propyleneglycol and polyethylene glycol. The injectable must be sterile and freeof pyrogens.

Nucleic acid molecules that encode FLAME-1 or FLAME-2 may be deliveredusing any one of a variety of delivery components, such as recombinantviral expression vectors or other suitable delivery means, so as toaffect their introduction and expression in compatible host cells. Ingeneral, viral vectors may be DNA viruses such as recombinantadenoviruses and recombinant vaccinia viruses or RNA viruses such asrecombinant retroviruses. Other recombinant vectors include recombinantprokaryotes which can infect cells and express recombinant genes. Inaddition to recombinant vectors, other delivery components are alsocontemplated such as encapsulation in liposomes, transferrin-mediatedtransfection and other receptor-mediated means. The invention isintended to include such other forms of expression vectors and othersuitable delivery means which serve equivalent functions and whichbecome known in the art subsequently hereto.

In one embodiment of the present invention, DNA is delivered tocompetent host cells by means of an adenovirus.

One skilled in the art would readily understand this technique ofdelivering DNA to a host cell by such means. Although the inventionpreferably includes adenovirus, the invention is intended to include anyvirus which serves equivalent functions.

In another embodiment of the present invention, RNA is delivered tocompetent host cells by means of a retrovirus. One skilled in the artwould readily understand this technique of delivering RNA to a host cellby such means. Any retrovirus which serves to express the proteinencoded by the RNA is intended to be included in the present invention.

In another embodiment of the present invention, nucleic acid isdelivered through folate receptor means. The nucleic acid sequence to bedelivered to a cell is linked to polylysine and the complex is deliveredto cells by means of the folate receptor. U.S. Pat. No. 5,108,921 issuedApr. 28, 1992 to Low et al., which is incorporated herein by reference,describes such delivery components.

Pharmaceutical compositions according to the invention include deliverycomponents in combination with nucleic acid molecules that encodeFLAME-1 or FLAME-2 which further comprise a pharmaceutically acceptablecarriers or vehicles, such as, for example, saline. Any medium may beused which allows for successful delivery of the nucleic acid. Oneskilled in the art would readily comprehend the multitude ofpharmaceutically acceptable media that may be used in the presentinvention.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the active agent to reach theagent's site of action in the body of a mammal. Pharmaceuticalcompositions may be administered parenterally, i.e., intravenous,subcutaneous, intramuscular. Intravenous administration is the preferredroute.

Dosage varies-depending upon known factors such as the pharmacodynamiccharacteristics of the particular agent, and its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms, kind of concurrent treatment, frequency oftreatment, and the effect desired.

According to one aspect of the invention, compounds may be screened toidentify FLAME-1 or FLAME-2 inhibitors, activators or compounds thatinterfere with or disrupt FLAME-1 or FLAME-2 interactions with Fas,TNFR1, FADD, Mch4 and Mch5. Inhibitors of FLAME-1 or FLAME-2 are usefulas apoptotic agents. Activators of FLAME-1 or FLAME-2 are useful asanti-apoptotic agents.

Ware, C. F. et al. 1996 J. Cell. Biochem. 60(1) :47-55, Nagata S. 1997Cell 88(3):355-65, Nagata S. 1996 Adv Exp Med Biol. 406:119-24, NagataS. and P. Golstein 1995 Science 267(5203):1449-56, Nagata S. 1994 AdvImmunol. 57:129-44, and Lu, M. L. et al. 1996 Proc. Natl. Acad. Sci. USA93(17):8977-82, which are each incorporated herein by reference eachdescribe Fas/TNFR1-induced apoptosis.

Nagata, S. 1997 Cell 88, 355-365 Rosette, C. and M. Karin, 1996 Science274, 1194-1197, which are both incorporated herein by reference eachdescribe UV-induced apoptosis.

Inhibitors of FLAME-1 or FLAME-2 are useful as apoptotic agents may beidentified by screening compounds to ascertain their effect on theanti-apoptosis activity of FLAME-1 or FLAME-2, respectively. In someembodiments of the invention, compounds are screened to identifyinhibitors by delivering FLAME-1 or FLAME-2 to cells in the presence orabsence of a test compound. Under assay conditions, the FLAME will havean anti-apoptotic effect on the cells in the absence of test compound.If in the presence of the test compound, the cells become apoptotic, thetest compound is candidate inhibitor of the FLAME. Antibodies whichinhibit FLAME activity are useful as inhibitors and, therefore aspositive controls in the assay. In some embodiments, the FLAME isdelivered to the cell as a protein. In some embodiments, the FLAME isdelivered to the cell as a nucleic acid molecule that encodes theprotein. In some embodiments of the invention, compounds are screened toidentify inhibitors by contacting the FLAME with a caspase moleculeknown to bind to the FLAME. The molecules are contacted in the presenceor absence of a test compound. Under assay conditions, the binding ofthe molecules in the absence of test compound but not in the presence ofthe compound indicates that the compound inhibits caspase/FLAME binding.Those having ordinary skill in the art can readily detect whether or notcaspase and FLAME molecules are bound to each other. Antibodies caninhibit FLAMEs from binding to caspase.

Activators of FLAME-1 or FLAME-2 are useful as anti-apoptotic agents maybe identified by screening compounds to ascertain their effect on theanti-apoptosis activity of FLAME-1 or FLAME-2, respectively. In someembodiments of the invention, compounds are screened to identifyactivators by delivering FLAME-1 or FLAME-2 to cells in the presence orabsence of a test compound. Under assay conditions, the cells will beapoptotic in the absence of test compound. If in the presence of thetest compound, the cells cease being apoptotic, the test compound iscandidate activator of the FLAME. In some embodiments, the FLAME isdelivered to the cell as a protein. In some embodiments, the FLAME isdelivered to the cell as a nucleic acid molecule that encodes theprotein.

The invention provides assays for screening compounds to identify andevaluating compounds that disrupt or interfere with FLAME interactionswith each other as well as Fas, FADD, TNFR1, Mch4 and Mch5 molecules.Assays are provided for identifying compounds that inhibit FLAME-1 orFLAME-2 binding to FADD, Mch4, Mch5, TNFR1 or Fas, comprising the stepsof performing a test assay by contacting the FLAME with Fas, FADD,TNFR1, Mch4 or Mch5 in the presence of a test compound under conditionsin which the FLAME binds to the Fas, FADD, TNFR1, Mch4 or Mch5 in theabsence of the test compound and determining whether the FLAME binds tothe Fas, FADD, TNFR1, Mch4 or Mch5. Assays are provided for identifyingcompounds that inhibit FLAME-1 binding to FLAME-2 comprising the stepsof performing a test assay by contacting the FLAME-1 with FLAME-2 in thepresence of a test compound under conditions in which the FLAME-1 bindsto the FLAME-2 in the absence of the test compound and determiningwhether the FLAME-1 binds to the FLAME-2.

In some embodiments of the invention, the preferred concentration oftest compound is between 1μM and 500μM. A preferred concentration is10μM to 100μM. In some preferred embodiments, it is desirable to use aseries of dilutions of test compounds.

Kits are included which comprise containers with reagents necessary toscreen test compounds. Such kits include FLAME-1 or FLAME-2 and/or anucleic acid molecule that encodes FLAME-1 or FLAME-2 and instructionsfor performing the assay. Kits may include cells, and may optionallyinclude antibodies as a control.

According to another aspect of the invention, transgenic animals,particularly transgenic mice, are generated. In some embodiments, thetransgenic animals according to the invention contain a nucleic acidmolecule which encodes FLAME-1 or FLAME-2. Such transgenic mice may beused as animal models for studying overexpression of FLAME-1 or FLAME-2and for use in drug evaluation and discovery efforts to find compoundseffective to inhibit or modulate the activity of FLAME-1 or FLAME-2. Onehaving ordinary skill in the art using standard techniques, such asthose taught in U.S. Pat. No. 4,873,191 issued Oct. 10, 1989 Wagner andU.S. Pat. No. 4,736,866 issued Apr. 12, 1988 to Leder, both of which areincorporated herein by reference, can produce transgenic animals whichproduce the FLAME-1 or FLAME-2 and use the animals in drug evaluationand discovery projects.

Another aspect of the present invention relates to knock-out mice andmethods of using the same. In particular, transgenic mice may begenerated which are homozygous for a mutated, non-functional FLAME-1and/or FLAME-2 gene which is introduced into them using well knowntechniques. The mice produce no functional FLAME-1 and/or FLAME-2 andare useful to study the function of FLAME-1 and/or FLAME-2. Furthermore,the mice may be used in assays to study the effect of test compounds onFLAME deficiency. The FLAME deficient mice can be used to determine if,how and to what extent FLAME inhibitors will effect the animal andthereby address concerns associated with inhibiting the activity of themolecule.

Methods of generating genetically deficient "knock out" mice are wellknown and disclosed in Capecchi, M. R. (1989) Science 244:1288-1292 andLi, P. et al. (1995) CELL 80:401-411, which are each incorporated hereinby reference. The human FLAME cDNA clone or the murine FLAME cDNA clonesuch as the murine FLAME-2 cDNA set forth in SEQ ID NO:5 can be used toisolate a murine FLAME genomic clone. The genomic clone can be used toprepare a FLAME targeting construct which can disrupt the FLAME gene inthe mouse by homologous recombination.

The targeting construct contains a non-functioning portion of the FLAMEgene which inserts in place of the functioning portion of the nativemouse gene. The non-functioning insert generally contains an insertionin the exon that encodes the active region of FLAME-1 or FLAME-2. Thetargeting construct can contain markers for both positive and negativeselection. The positive selection marker allows for the selectiveelimination of cells without it while the negative selection markerallow; for the elimination of cells that carry it.

For example, a first selectable marker is a positive marker that willallow for the survival of cells carrying it. In some embodiments, thefirst selectable marker is an antibiotic resistance gene such as theneomycin resistance gene can be placed within the coding sequences ofthe Mch2 gene to render it non-functional while additionally renderingthe construct selectable. The antibiotic resistance gene is within thehomologous region which can recombine with native sequences. Thus, uponhomologous reconstruction, the non-functional and antibiotic resistanceselectable gene sequences will be taken up.

The targeting construct also contains a second selectable marker whichis a negative selectable marker. Cells with the negative selectablemarker will be eliminated. The second selectable marker is outside therecombination region. Thus, if the entire construct is present in thecell, both markers will be present. If the construct has recombined withnative sequences, the first selectable marker will be incorporated intothe genome and the second will be lost. The herpes simplex virusthymidine kinase (HSV tk) gene is an example of a negative selectablemarker which can be used as a second marker to eliminate cells thatcarry it. Cells with the HSV tk gene are selectively killed in thepresence of gangcyclovir.

Cells are transfected with targeting constructs and then selected forthe presence of the first selection marker and the absence of thesecond. Clones are then injected into the blastocysts and implanted intopseudopregnant females. Chimeric offspring which are capable oftransferring the recombinant genes in their germline are selected, matedand their offspring is examined for heterozygous carriers of therecombined genes. Mating of the heterozygous offspring can then be usedto generate fully homozygous offspring which are the FLAME-deficientknock out mouse.

The present invention relates to methods of and compositions forinhibiting the expression of FLAME-1 or FLAME-2 in cells. In oneembodiment, antisense oligonucleotides are provided which have anucleotide sequence complementary to a nucleotide sequence of mRNA thatencodes FLAME-1 or FLAME-2.

The antisense oligonucleotides of the present invention comprisesequences complementary to regions of FLAME-1 or FLAME-2 mRNA. Theoligonucleotides comprise a sequence complementary to a region selectedfrom the sequence of the FLAME mRNA. The antisense oligonucleotidesinclude single stranded DNA sequence and an antisense RNAoligonucleotide produced from an expression vector. Each of theantisense oligonucleotides of the present invention are complementary toregions of the FLAME mRNA sequence.

The antisense oligonucleotides of the present invention comprises asequence complementary to a fragment of SEQ ID NO:1 or SEQ ID NO:3. SeeUllrich et al., EMBO J., 1986, 5:2503, which is incorporated herein byreference. Contemplated by this definition are fragments of oligoswithin the coding sequence for FLAME-1 or FLAME-2. Oligonucleotides arepreferably complementary to a nucleotide sequence that is 5-50nucleotides in length, in some embodiments 8-40, more preferably 12-25nucleotides, in some embodiments 10-15 nucleotides and in someembodiments 12-20 nucleotides.

In addition, mismatches within the sequences identified above, whichachieve the methods of the invention, such that the mismatched sequencesare substantially complementary to the FLAME sequences are alsoconsidered within the scope of the disclosure. Mismatches which permitsubstantial complementarily to the FLAME sequences will be known tothose of skill in the art once armed with the present disclosure. Theoligos may also be unmodified or modified.

The present invention is also directed to a method of inhibiting FLAME-1or FLAME-2 expression in mammals comprising contacting the mammal withan effective amount of an antisense oligonucleotide having a sequencewhich is complementary to a region of the FLAME-1 mRNA or FLAME-2 mRNA,respectively.

Methods of administering the antisense oligos of the present inventioninclude techniques well known in the art such as and not limited toliposomes, plasmid expression, or viral vector including retroviralvectors. In the administration of oligos via vectors or plasmids, anon-coding RNA strand of FLAME is preferably used in order to produceantisense RNA oligos which are expressed by the cell. The RNA oligosthen bind FLAME sense or coding RNA sequence.

Methods of administering the oligos to mammals include liposomes, andmay be in a mixture with a pharmaceutically-acceptable carrier, selectedwith regard to the intended route of administration and the standardpharmaceutical practice. In addition, antibodies, ligands and the likemay be incorporated into the liposomes thereby providing various modesof inhibiting FLAME expression. Dosages will be set with regard toweight, and clinical condition of the patient. The proportional ratio ofactive ingredient to carrier will naturally depend on the chemicalnature, solubility, and stability of the compounds, as well as thedosage contemplated. The oligos of the present invention will beadministered for a time sufficient for the mammals to be free ofundifferentiated cells and/or cells having an abnormal phenotype.

The oligos of the invention may be employed in the method of theinvention singly or in combination with other compounds. The amount tobe administered will also depend on such factors as the age, weight, andclinical condition of the patient. See Gennaro, Alfonso, ed.,Remington's Pharmaceutical Sciences, 18th Edition, 1990, Mack PublishingCo., Easton Pa.

The compounds of the present invention may be administered by anysuitable route, including inoculation and injection, for example,intravenous, oral, intraperitoneal, intramuscular, subcutaneous,topically, and by absorption through epithelial or mucocutaneouslinings, for example, nasal, oral, vaginal, rectal and gastrointestinal.

The mode of administration of the oligos may determine the sites in theorganism to which the compound will be delivered. For instance, topicalapplication may be administered in creams, ointments, gels, oils,emulsions, pastes, lotions, and the like. The oligos of the presentinvention may be administered alone or will generally be administered inadmixture with a pharmaceutical carrier selected with regard to theintended route of administration and standard pharmaceutical practice.For parenteral administration, they are best used in the form of sterileaqueous solution which may contain other solutes, for example,sufficient salts, glucose or dextrose to make the solution isotonic. Fororal mode of administration, the present invention may be used in theform of tablets, capsules, lozenges, troches, powders, syrups, elixirs,aqueous solutions and suspension, and the like. Various disintegrantssuch as starch, and lubricating agents may be used. For oraladministration in capsule form, useful diluents are lactose and highmolecular weight polyethylene glycols. When aqueous suspensions arerequired for oral use, certain sweetening and/or flavoring agents may beadded. Forty μg/ml antisense oligo was used for in vitro methods ofproviding oligos in media for cell growth in culture. This concentrationmay be extrapolated for in vivo use. The concentration of antisenseoligonucleotides for in vivo use is about 40μ/g body weight. The in vivouse of the expression vector expressing RNA oligonucleotides isdetermined by the number of transfected cells.

For in vivo use, the antisense oligonucleotide may be combined with apharmaceutically acceptable carrier, such as suitable liquid vehicle orexcipient and an optional auxiliary additive or additives. The liquidvehicles and excipients are conventional and commercially available.Illustrative thereof are distilled water, physiological saline, aqueoussolution of dextrose, and the like. For in vivo antineoplastic use, theantisense oligonucleotides may be administered intravenously.

In addition to administration with conventional carriers, antisenseoligonucleotides may be administered by a variety of specializedoligonucleotide delivery techniques. For example, oligonucleotides havebeen successfully encapsulated in unilamellar liposomes. ReconstitutedSendai virus envelopes have been successfully used to deliver RNA andDNA to cells. Arad et al., Biochem. Biophy. Acta., 1986, 859, 88-94.

EXAMPLE

In order to characterize novel FADD-like apoptotic/anti-apoptoticmolecules (FLAMEs) that interact with caspases, the Genbank expressedsequence tags (ESTs) data base was searched for sequences that arehomologous to the FADD-like caspases Mch4 (caspase-10) andMch5/MACH/FLICE (caspase-8) (Fernandes-Alnemri, T. et al., 1996 Proc.Natl. Acad. Sci. USA. 93, 7464-7469, Boldin, M. P. 1996 Cell 85,803-815, and Muzio, M. et al. 1996 Cell 85, 817-827, which are eachincorporated herein by reference). Two ESTs (clones 427786 and 576731)with statistically significant similarity to Mch5 (p<0.001) wereidentified. The 5' sequence of EST clone 427786 suggested that it is a5'-truncated complementary DNA (cDNA) clone. PCR primers weresynthesized and used to amplify a partial cDNA probe. The full lengthFLAME-1 cDNA was cloned from human Jurkat Uni-ZAP XR cDNA library(Fernandes-Alnemri, T. et al. 1994 J. Biol. Chem. 269, 30761, which isincorporated herein by reference) by screening the library with apartial FLAME-1 cDNA probe. The probe was amplified from Jurkat cDNAlibrary by two PCR amplification steps using FLAME-1 specific primersderived from the 3' (Genbank accession # aa002262, which is incorporatedherein by reference) and 5' (Genbank accession # aa001257, which isincorporated herein by reference) sequences of human EST clone 427786.The primary PCR was done with Mchx-pr1 (AGGCTGGTCTCGAACTCC--SEQ ID NO:7)and Mchx-pr3 (TTCTCCAAGCAGCAATCC--SEQ ID NO:8). The secondary PCR wasdone with Mchx-pr2 (GGCCTCCCAAAGTGCTGG--SEQ ID NO:9) and Mchx-pr4(TTCAGGCTCCATAATGGG--SEQ ID NO:10). The PCR product was cloned into SmaI site of pBluescript II KS+ vector and then used to screen the JurkatcDNA library. The beta-isoform of FLAME-1 was cloned by RT-PCR.

This probe was used to isolate and clone the full length cDNA (˜1.9 kb)from a human Jurkat cDNA library, that encodes a novel protein(designated FLAME-1) of 445 amino acids with predicted relativemolecular mass of 51 kDa (SEQ ID NO: 1; FIG. 1A). FIG. 1A showsco-linear alignment of the predicted amino acid sequence of humanFLAME-1 with proMch4 and proMch5b and a schematic diagram of itsstructure. Based on the crystal structure of ICE and CPP32, the residuesmarked with a (c) are involved in catalysis and those marked with a (b)are involved in binding the carboxylate side chain of the substrate P1aspartate. The active site pentapeptide QACQG (SEQ ID NO:11) in Mch4 andMch5 is boxed. The residues that are unique to FLAME-lb are underlined.The vertical arrow indicates the splice junction, after which FLAME-lbdiffers from FLAME-1.

FLAME-1 is most similar to the Mch4 and Mch5 caspases. It has threedistinct homology regions: Two N-terminal tandem stretches ofapproximately 67-79 residues that are significantly homologous to theN-terminal DED (residues 1-79) of FADD, here referred to as FADD-DEDhomology A (FDH-A, residues 5-71) and B (FDH-B, residues 90-168)regions. FDH-A and FDH-B share 38% and 28% identity with the DED ofFADD, respectively. The FDH regions share 28-33% identity with thecorresponding regions in Mch4 and Mch5b. The FDH regions are followed bya stretch of 249 residues (residues 197-445) that is significantlyhomologous to the region which encodes the large and small subunits ofknown caspases, here referred to as the caspase-domain homology (CDH)region. Although this region shares approximately 27-31% identity withthe corresponding regions in Mch4 and Mch5b, there are severaldifferences. This region contains a QNYVV (SEQ ID NO:12) motif insteadof the conserved active site motif QACXG (X=R,Q,G--SEQ ID NO:13, SEQ IDNO:11, SEQ ID NO:14, respectively), present in caspases. Also, only one(G281) out of the three residues involved in catalysis, and two (Q323and S386) out of the four residues involved in binding the carboxylateside chain of the substrate P1 aspartate, are conserved. This regioncontains a potential caspase cleavage site (LEVD-G--ID NO:15) C-terminalto the QNYVV (SEQ ID NO:12) motif, that can be cleaved by caspases togenerate two polypeptides (p39 and p12) corresponding to the large andsmall subunits of caspases. These observations suggest that FLAME-1could be a protease with a different substrate specificity compared tocaspases, or an enzymatically inactive protein. Because of the presenceof CDH and FDH regions, FLAME-1 would be predicted to interact withcaspases and/or other FDH-containing proteins. A naturally existingalternatively spliced isoform of FLAME-1 (FLAME-1b) lacking the entireCDH region was also identified by RT-PCR. This isoform shares residues1-231 with FLAME-1 but has a 39 amino acid-long unique C-terminus.

Clone 576731 contained a consensus Kozak translation initiation ATGcodon, preceded by a stop codon (12 bp upstream), characteristic of afull length cDNA clone of FLAME-2 (SEQ ID NO:3; FIG. 1B). FIG. 1B showsthe predicted amino acid sequence of FLAME-2 and its structure. TheIMAGE Consortium clone 576731, was characterized by automated sequencingand found to encode full length FLAME-2. This clone was then used toscreen the EST data base to identify the human counterpart. Severalhuman ESTs were identified and their sequence information was used todesign primers corresponding to the first and last six amino acids ofhuman FLAME-2. Human FLAME-2 was then cloned by PCR from JurkatT-lymphocytes. A BLAST search of the EST data base identified severaloverlapping human and mouse clones encoding the same protein. The humanand mouse cDNAs (˜2.2 kb) encode a novel protein (designated FLAME-2) of318 amino acids with predicted relative molecular mass of 37 kDa. Thehuman and mouse counterparts (proteins) are 99.3% identical. The onlydifference between the human and mouse FLAME-2 counterparts areunderlined in FIG. 1B (human/mouse, R/K, P/S, A/T).

FIG. 1C shows the N-terminal region of FLAME-2 (amino acids 23-101)shares significant homology with the FDH-A of Mch5b and the N-terminalDED of FADD. FLAME-2 has a similar organization to FADD. It has anN-terminal FDH region (residues 23-101) that shares ˜22% identity withthe FADD-DED region (residues 1-79) and 20-30% identity with the FDHregions of Mch4, Mch5 and FLAME-1. However, its C-terminal domain (CTD,residues 102-318) is unique in that it shares no significant homologywith the CTD of FADD (also known as the death domain) or any other knownproteins. The structure of FLAME-2 suggests that it could be an adaptormolecule for an as yet unidentified signaling complex.

To determine the distribution of FLAME-1 and 2, various tissue mRNAsamples were subjected to Northern blot analysis. Tissue distributionanalysis of FLAME-1 and FLAME-2 mRNAs was performed on Northern blotsprepared by Clontech containing 2 μg/lane of poly A+ RNA. Radioactiveriboprobes were prepared by using human FLAME-1-CDH or FLAME-2-FDH cDNAsas templates for SP6 RNA polymerase in the presence of [a32P] CTP. Theblots were hybridized, washed and then visualized by autoradiography.Numbers on the right indicate kilobases. PBLs, peripheral bloodleukocyte. As shown in FIG. 1D, FLAME-1 mRNA (˜1.9 kb) is expressedmainly in testes and skeletal muscle. This message is less abundant inthe other human tissues examined. However, a -1.2 kb abundant message isexpressed in the placenta, which could be an alternatively splicedisoform of FLAME-1 mRNA. FLAME-2 mRNA (˜2.2 kb), on the other hand ismore abundant than FLAME-1 mRNA. It is constitutively expressed in allthe tissues examined with particularly high expression in testes,skeletal muscle, heart and placenta.

Chromosomal mapping linked the FLAME-1 and Mch5 genes to the D2S116 andD2S348 markers on chromosome 2q33-34 using radiation hybrid panels, inclose proximity to where they were previously localized Mch4. The humangenes for Mch5, FLAME-1 and FLAME-2 were mapped on a previouslydescribed rodent-human hybrid panels (Bullrich F. et al., 1995 CancerRes. 55, 1199) and on the Genebridge 4 and Stanford G3 radiation hybridpanels (Research Genetics) using specific oligonucleotide primers.Radiation hybrid scoring data were submitted to the Whitehead Institute(WI) (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper), andStanford (http://shgc-www.stanford.edu) radiation hybrid servers. Dataavailable through public databases and published genome maps (ChumakovI. et al., 1995 Nature 377.supp. 175) was then used to confirm thephysical localization of markers and genes. As shown in FIG. 1E, FLAME-2is localized to chromosome 1q23-24. This finding and the high degree ofhomology among their genes or gene products suggest that they might bedescendents of a common ancestral gene through gene duplication. Thisfinding is important because genetic lesions in this locus may havedramatic effects on Fas/TNFR1-induced apoptosis. The FLAME-2 gene wasmapped to chromosome 1 within 6.51 cR of the CHLC.GATA43A04 marker in a16 cM region between the D1S305 and D1S445 markers at 1q23-24.

To determine whether FLAME-1 possesses caspase activity, C-terminalHis-tagged full length or truncated FLAME-1 lacking the FDH regions wereexpressed in bacteria or in the baculovirus expression system. UnlikeMch4 or Mch5, expression of these constructs did not result in cleavageof FLAME-1 (autoactivation) or generation of a caspase-like activity asdetermined with the tetrapeptide substrates YVAD-AMC (SEQ ID NO:16) orDEVD-AMC (SEQ ID NO:17), suggesting that FLAME-1 might be enzymaticallyinactive or possess an unknown enzymatic activity.

As shown in FIG. 2A, in vitro translated FLAME-1 can be cleaved byseveral caspases including CPP32, Mch2, Mch3, Mch4, Mch5 and ICH-1 togenerate two fragments (p39 and p12) topologically equivalent to thelarge and small subunits of caspases. This cleavage occurs at Asp341 inthe LEVD-G site, since a D to A mutation in this site prevents thesecaspases from cleaving FLAME-1. In the experiments shown in FIG. 2A,FLAME-1 or FLAME-1-D341A were 35S-labeled in vitro using Promega's TNTkit (Srinivasula, S. M. et al. 1996 Proc. Natl.

Acad. Sci. USA. 93, 13706-13711 and Ahmad, M. et al. 1997 Cancer. Res.57, 615-619). The labeled proteins were incubated with 100 ng ofpurified recombinant caspases and then analyzed as described before. Invitro translated FLAME-1 (upper panel) or FLAME-1-D341A (lower panel)were incubated without (lane 1) or with caspase-2 (ICH-1), caspase-3(CPP32), caspase-6 (Mch2), caspase-7 (Mch3), caspase-8 (Mch5) orcaspase-10 (Mch4) (lanes 2-7, respectively) for 1 h at 370C. Proteinswere then analyzed by SDS-PAGE and autoradiography. Full length FLAME-1,and its p39 and p12 fragments are indicated to the right.

Transfection studies showed that FLAME-1 may also be a caspase substratein vivo. Expression of a T7-epitope tagged FLAME-1 (T7-FLAME-1) in 293cells produced both full length and cleaved (p39) FLAME-1 (See FIGS. 3Band 3F). This cleavage was not observed with the D341A mutant FLAME-1(T7-FLAME-1-D341A, see FIGS. 3C and 3F). Furthermore, stimulation ofFLAME-1-transfected MCF7-FAS cells with anti-Fas antibody increased theamount of cleavage products. Thus, FLAME-1 appears to be a caspasetarget in apoptotic cells.

To investigate the participation of FLAME-1 and FLAME-2 in Fas/TNFR1apoptotic signaling pathways, in vitro and in vivo binding studies andyeast-two hybrid analysis were performed and the data is shown in FIGS.2B-2E. Radiolabeled FLAME-1, Mch4, Mch5b, FADD or mutants of theseproteins were precipitated with various glutathione-S-transferase (GST)fusion proteins immobilized on glutathione-Sepharose beads. Constructsencoding GST fusion proteins were prepared using the bacterialexpression vector pGEX-2T. The GST-fusion proteins were expressed inDH5a bacteria and then immobilized on glutathione-Sepharose. Labeledinteracting proteins were prepared by in vitro transcription andtranslation in the presence of 35S-[methionine]. Following translation,equivalent amounts of the labeled proteins were incubated with variousimmobilized GST-fusion proteins. The beads were washed and boiled inSDS-sample buffer. The eluted proteins were resolved by SDS-PAGE andvisualized by autoradiography. The indicated in vitro translated35S-labeled proteins were precipitated with GST, (lanes 1b-e) orGST-FADD (lanes 2b), GST-FLAME-1-beta (lanes 2c), GST-FLAME-1-CDH(residues 196-445) (lanes 2d) or GST-FLAME-2 (lanes 2e) fusion proteinsimmobilized on glutathione-Sepharose beads. The bound proteins were thenanalyzed by SDS-PAGE and autoradiography. Truncated proteins FADD-DED orFADD-DD contain residues 1-79 or 80-205, respectively. The data in FIGS.2B-2E shows that Mch4, Mch5b and FLAME-1 associated specifically withGST-FADD, although the interaction of FLAME-1 with FADD was weaker thanthat observed with Mch4 or Mch5b. FADD, FADD-DED, Mch4, Mch5b, andMch5b-FDH, but not FADD-DD, also associated specifically with FLAME-1b(GST-FLAME-1-beta). These observations suggest that the interactions aremediated by the homologous FDH regions of these proteins. Mch5b but notMch4 associated with a truncated FLAME-1 lacking its FDH regions(GST-FLAME-1-CDH), suggesting that the two proteins can also interactthrough their homologous CDH regions. GST-FLAME-2 associated stronglywith FLAME-1 and weakly with Mch4 or Mch5b, but did not associate withFADD.

To demonstrate these interactions in vivo, 293 cells were transientlytransfected with plasmids encoding T7-epitope tagged FADD, FLAME-1,FLAME-2 or mutants and various Flag epitope-tagged proteins. Becausewild type Mch4, Mch5b and their CDH regions are potent inducers ofapoptosis in 293 cells, active site Cys to Ala Flag-tagged mutants wereused in these experiments to investigate their interactions with FLAME-1and FLAME-2. FIGS. 3A-3F show FLAME-1 coimmunoprecipite with FADD, Mch4,Mch5b, and FLAME-2. 293 cells were transfected with expression plasmidsencoding T7-epitope tagged FADD (FIG. 3A), FLAME-1 (FIG. 3B),FLAME-1-D341A (FIG. 3C), FLAME-1-CDH (residues 196-445) (FIG. 3D) orFLAME-2 (FIG. 3E), and different Flag-epitope tagged proteins asfollows: 0, no Flag-plasmid; 1, FLAME-1; 2, FLAME-lb; 3, FLAME-2; 4,FLAME-2-FDH (residues 1-106); 5, Mch5b C345A; 6, Mch5b-FDH (residues1-201); 7, Mch4 C358A; 8, Mch4-FDH (residues 18-189); 9, FADD; 10,Mch5b-CDH C345A (residues 201-464); 11, Mch4-CDH C358A (residues200-479). A modified expression vector (T7-pcDNA3) encoding a T7-epitopetag under the CMV promoter was constructed by subcloning the T7-tagcoding sequence of pET21b (Invitrogen) into the EcoRV site of pcDNA3.Epitope tagging was done by cloning cDNAs inframe into the multiplecloning sites of T7-pcDNA3 and/or the Flag plasmid pFLAG-CMV-2 (IBIKodak). N-terminal and C-terminal deletion mutants were generated byPCR. Point mutants were generated by site directed mutagenesis usingoverlapping PCR mutagenic oligonucleotides. All PCR products wereverified by sequencing. cDNAs of FLAME-1 or FLAME-2 without epitope tagswere subcloned into pcDNA3. Flag-tagged Fas was constructed in pcDNA3.After 34-36 h, extracts were prepared and immunoprecipitated with amonoclonal antibody to the Flag-epitope. 293 or 293T human embryonickidney cells were transiently transfected with the expression plasmidsusing the LipofectAMINE (Life Technologies) method. Cells were lysed ina lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% NP-40) andincubated with anti Flag-M5 monoclonal antibody (IBI Kodak). The immunecomplexes were precipitated with protein-A/G-Sepharose, washed and theneluted by boiling in SDS-sample buffer. The eluted proteins wereresolved by SDS-PAGE and detected by Western analysis with aHRP-conjugated T7-antibody (Novagen). The samples were analyzed bySDS-PAGE and Western blotted with a horseradish peroxidase(HRP)-conjugated T7-antibody. All extracts were immunoblotted withanti-Flag and anti-T7 to verify expression of the encoded proteins. FIG.3F shows FLAME-1 is recruited to the Fas signaling complex. 293T cellswere transfected with the indicated expression plasmids,immunoprecipitated and detected as in FIGS. 3A-3D.

Consistent with the in vitro results, T7-FADD coprecipitated with fulllength FLAME-1, Mch4 and Mch5b, or their isolated FDH regions, but notwith FLAME-2 or FLAME-2-FDH (FIG. 3A). T7-FLAME-1 and its p39 fragmentcoprecipitated with full length FADD, Mch4, Mch5b or their isolated FDHregions (FIG. 3B). Full length T7-FLAME-1, but negligible amount of thep39 fragment, associated with Mch5-CDH, FLAME-2 or FLAME-2 FDH,suggesting that the entire CDH region of FLAME-1 is required for optimalinteraction between these proteins. Similar results were obtained withT7-FLAME-1-D341A and T7-FLAME-1-CDH (FIGS. 3C and 3D). No interactionswere observed between T7-FLAME-lb and Flag-Mch4-CDH or Flag-Mch5b-CDH,suggesting that these proteins can only interact through theirrespective FDH or CDH regions. Also consistent with the in vitro data,T7-FLAME-2 interacted with Mch4 and Mch5b (FIG. 3E). The yeast-twohybrid analysis confirmed the interactions of FLAME-1-FDH with FADD,Mch4 and Mch5 FDH regions (Table 1). Mch4-FDH (residues 18-189), Mch5b(FDH A, residues 3-80; FDH B, residues 102-177; FDH, residues 3-177),FLAME-1-FDH (residues 1-160), and murine FADD-DED (residues 1-78) weresubcloned into yeast two-hybrid vectors. Yeast-two hybrid analysis wasthen performed. This analysis also revealed that FLAME-1-FDH can alsostrongly interact with itself (Table 1).

FADD can recruit Mch5 (MACH/FLICE) and possibly Mch4 to the Fas/TNFR1signaling complex. To determine whether FLAME-1 can also be recruitedthrough FADD, coprecipitation experiments were performed in 293T cells(FIG. 3F). FLAME-1 or FLAME-1-D341A were able to form a complex with Fas(lanes 4 and 8), possibly through interaction with endogenous FADD.Cotransfection of exogenous T7-FADD enhanced the FLAME-1-Fas interaction(lanes 5 and 7). The p39 fragment which is generated by cleavage atAsp341 also formed a complex with Fas (lane 5). These observationsdemonstrate that FLAME-1 can be recruited to a Fas signaling complexand, thus, may participate in the Fas signaling pathway. FLAME-2, on theother hand, did not form a complex with Fas in the presence or absenceof exogenous FADD. However, it is still possible that FLAME-2 couldinteract with the Fas-death complex through other molecules that mightbe limited in the cell, such as Mch4 or Mch5.

To study the functional role of FLAME-1 or 2 in Fas/TNFR1- or UV-inducedapoptosis, they were transfected into MCF-7-FAS cells. FIGS. 4A and 4Bshow MCF7-FAS cells were transfected with the indicated expressionplasmids. Cells were treated 28 h after transfection with eitheranti-Fas antibody, TNF, or UV-irradiation. MCF7-FAS cells weretransiently cotransfected with reporter and test plasmids at a ratio of1:10 and assayed for apoptosis. The percentage of viable cells (mean±SD) under each condition was determined by measuring the number ofviable blue cells compared with total blue cells. Control cells receivedno treatment. The cells were fixed, stained for b-GAL expression andthen viewed by phase-contrast microscopy. Neither FLAME-1 nor 2 inducedapoptosis in these cells. However, FLAME-1 and FLAME-1b significantlyblocked Fas- and TNFR1-induced apoptosis but not UV-induced apoptosis(FIG. 1A). This indicates that overexpression of the FDH regions ofFLAME-1 is sufficient to block Fas/TNFR1-induced apoptosis. Thisprotective effect approached 60-65% of that observed by Bcl-xLoverexpression. FLAME-2, on the other hand, effectively blockedUV-induced apoptosis to a level approaching that observed byoverexpression of baculovirus p35, and to a lesser degree inhibitedTNFR1-induced apoptosis. Slight inhibition of Fas-induced apoptosis wasalso observed.

Taken together, the data presented here establish FLAME-1 and -2 as thefirst examples of endogenous FDH-containing proteins which can act asnegative regulators of apoptosis. Recently, viral FDH-containingproteins E8 and MC159 were demonstrated to abrogate Fas/TNFR1 mediatedapoptosis (Bertin, J. et al., 1997 Proc. Natl. Acad. Sci. USA. 94,1172-1176). Both FLAME-1 and the viral proteins appear to target theFas/FADD/caspase signaling complex by a potential dominant negativemechanism. Binding of FLAME-1, its FDH regions or the viral proteins tothe caspase Mch4 or Mch5 or the adaptor molecule FADD blocksFas/TNFR1-induced apoptosis possibly by interfering with the assembly ofa functional death receptor signaling complex. In contrast to FLAME-1and the viral proteins, FLAME-2 significantly abrogated UV-inducedapoptosis. Recently, UV stimulation of cells was shown to lead to theactivation of cell surface TNF receptors. Although FLAME-2 doessignificantly inhibit TNF mediated death, a purely TNF mediatedUV-induced cell death is not supported here; This is because FLAME-1possesses significant inhibitory activity towards TNF-induced death, yetit has no anti-apoptotic activity against UV. These results suggest thatUV-induced apoptosis may be mediated by a novel FDH-containing adaptormolecule(s), which may be the target(s) for FLAME-2 anti-apoptoticactivity. Consequently, it appears that molecules which contain FDHregions could be either pro-apoptotic like FADD, Mch4 or Mch5, oranti-apoptotic such as FLAME-1, FLAME-2 and the viral proteins E8 andMC159. Since the pro-apoptotic or anti-apoptotic proteins might havedifferent expression levels, their ratios could determines how a givencell or cell type respond to FasL, TNF or UV. Targeted knockout of thesemolecules should help understand their exact role in apoptosis and otherbiological processes.

                  TABLE 1                                                         ______________________________________                                        FADD Domain Homology (FDH) interactions by the yeast                           two-hybrid assay.                                                                                            Liquid assay                                                                     b-gal activity,                              DNA-binding hybrid Activation hybrid Miller units                           ______________________________________                                        LeXA-FLAME-1-FDH                                                                           B42            23.2 ±                                                                              0.8                                        LeXA-FLAME-1-FDH B42-Mch5b-FDH-A 52.7 ± 22.6                               LeXA-FLAME-1-FDH B42-Mch5b-FDH-B 728.1 ± 38.5                              LeXA-FLAME-1-FDH B42-Mch5b-FDH 300.2 ± 42.3                                LeXA-FLAME-1-FDH B42-Mch4-FDH 697.6 ± 103.6                                LeXA-FLAME-1-FDH B42-FADD-DED 324.1 ± 34.0                                 LeXA-FLAME-1-FDH B42-FLAME-1-FDH 1634.0 ± 297.0                          ______________________________________                                         Data represent four independent experiments.                             

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  17                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1750 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: both                                                   - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 413..1750                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CGAGTCTCAA CTAAAAGGGA CTCCCGGAGC TAGGGGTGGG GACTCGGCCT CA -            #CACAGTGA     60                                                                 - - GTGCCGGCTA TTGGACTTTT GTCCAGTGAC AGCTGAGACA ACAAGGACCA CG -            #GGAGGAGG    120                                                                 - - TGTAGGAGAG AAGCGCCGCG AACAGCGATC GCCCAGCACC AAGTCCGCTT CC -            #AGGCTTTC    180                                                                 - - GGTTTCTTTG CCTCCATCTT GGGTGCGCCT TCCCGGCGTC TAGGGGAGCG AA -            #GGCTGAGG    240                                                                 - - TGGCAGCGGC AGGAGAGTCC GGCCGCGACA GGACGAACTC CCCCACTGGA AA -            #GGATTCTG    300                                                                 - - AAAGAAATGA AGTCAGCCCT CAGAAATGAA GTTGACTGCC TGCTGGCTTT CT -            #GTTGACTG    360                                                                 - - GCCCGGAGCT GTACTGCAAG ACCCTTGTGA GCTTCCCTAG TCTAAGAGTA GG - # ATG            415                                                                                        - #                  - #                  - #    Met                          - #                  - #                  - #      1         - - TCT GCT GAA GTC ATC CAT CAG GTT GAA GAA GC - #A CTT GAT ACA GAT GAG          463                                                                       Ser Ala Glu Val Ile His Gln Val Glu Glu Al - #a Leu Asp Thr Asp Glu                         5    - #              10    - #              15                  - - AAG GAG ATG CTG CTC TTT TTG TGC CGG GAT GT - #T GCT ATA GAT GTG GTT          511                                                                       Lys Glu Met Leu Leu Phe Leu Cys Arg Asp Va - #l Ala Ile Asp Val Val                    20         - #         25         - #         30                      - - CCA CCT AAT GTC AGG GAC CTT CTG GAT ATT TT - #A CGG GAA AGA GGT AAG          559                                                                       Pro Pro Asn Val Arg Asp Leu Leu Asp Ile Le - #u Arg Glu Arg Gly Lys                35             - #     40             - #     45                          - - CTG TCT GTC GGG GAC TTG GCT GAA CTG CTC TA - #C AGA GTG AGG CGA TTT          607                                                                       Leu Ser Val Gly Asp Leu Ala Glu Leu Leu Ty - #r Arg Val Arg Arg Phe            50                 - # 55                 - # 60                 - # 65       - - GAC CTG CTC AAA CGT ATC TTG AAG ATG GAC AG - #A AAA GCT GTG GAG ACC          655                                                                       Asp Leu Leu Lys Arg Ile Leu Lys Met Asp Ar - #g Lys Ala Val Glu Thr                            70 - #                 75 - #                 80              - - CAC CTG CTC AGG AAC CCT CAC CTT GTT TCG GA - #C TAT AGA GTG CTG ATG          703                                                                       His Leu Leu Arg Asn Pro His Leu Val Ser As - #p Tyr Arg Val Leu Met                        85     - #             90     - #             95                  - - GCA GAG ATT GGT GAG GAT TTG GAT AAA TCT GA - #T GTG TCC TCA TTA ATT          751                                                                       Ala Glu Ile Gly Glu Asp Leu Asp Lys Ser As - #p Val Ser Ser Leu Ile                   100          - #       105          - #       110                      - - TTC CTC ATG AAG GAT TAC ATG GGC CGA GGC AA - #G ATA AGC AAG GAG AAG          799                                                                       Phe Leu Met Lys Asp Tyr Met Gly Arg Gly Ly - #s Ile Ser Lys Glu Lys               115              - #   120              - #   125                          - - AGT TTC TTG GAC CTT GTG GTT GAG TTG GAG AA - #A CTA AAT CTG GTT GCC          847                                                                       Ser Phe Leu Asp Leu Val Val Glu Leu Glu Ly - #s Leu Asn Leu Val Ala           130                 1 - #35                 1 - #40                 1 -      #45                                                                              - - CCA GAT CAA CTG GAT TTA TTA GAA AAA TGC CT - #A AAG AAC ATC CAC        AGA      895                                                                    Pro Asp Gln Leu Asp Leu Leu Glu Lys Cys Le - #u Lys Asn Ile His Arg                          150  - #               155  - #               160              - - ATA GAC CTG AAG ACA AAA ATC CAG AAG TAC AA - #G CAG TCT GTT CAA GGA          943                                                                       Ile Asp Leu Lys Thr Lys Ile Gln Lys Tyr Ly - #s Gln Ser Val Gln Gly                       165      - #           170      - #           175                  - - GCA GGG ACA AGT TAC AGG AAT GTT CTC CAA GC - #A GCA ATC CAA AAG AGT          991                                                                       Ala Gly Thr Ser Tyr Arg Asn Val Leu Gln Al - #a Ala Ile Gln Lys Ser                   180          - #       185          - #       190                      - - CTC AAG GAT CCT TCA AAT AAC TTC AGG AGC AT - #A CCT GAA GAG AGA TAC         1039                                                                       Leu Lys Asp Pro Ser Asn Asn Phe Arg Ser Il - #e Pro Glu Glu Arg Tyr               195              - #   200              - #   205                          - - AAG ATG AAG AGC AAG CCC CTA GGA ATC TGC CT - #G ATA ATC GAT TGC ATT         1087                                                                       Lys Met Lys Ser Lys Pro Leu Gly Ile Cys Le - #u Ile Ile Asp Cys Ile           210                 2 - #15                 2 - #20                 2 -      #25                                                                              - - GGC AAT GAG ACA GAG CTT CTT CGA GAC ACC TT - #C ACT TCC CTG GGC        TAT     1135                                                                    Gly Asn Glu Thr Glu Leu Leu Arg Asp Thr Ph - #e Thr Ser Leu Gly Tyr                          230  - #               235  - #               240              - - GAA GTC CAG AAA TTC TTG CAT CTC AGT ATG CA - #T GGT ATA TCC CAG ATT         1183                                                                       Glu Val Gln Lys Phe Leu His Leu Ser Met Hi - #s Gly Ile Ser Gln Ile                       245      - #           250      - #           255                  - - CTT GGC CAA TTT GCC TGT ATG CCC GAG CAC CG - #A GAC TAC GAC AGC TTT         1231                                                                       Leu Gly Gln Phe Ala Cys Met Pro Glu His Ar - #g Asp Tyr Asp Ser Phe                   260          - #       265          - #       270                      - - GTG TGT GTC CTG GTG AGC CGA GGA GGC TCC CA - #G AGT GTG TAT GGT GTG         1279                                                                       Val Cys Val Leu Val Ser Arg Gly Gly Ser Gl - #n Ser Val Tyr Gly Val               275              - #   280              - #   285                          - - GAT CAG ACT CAC TCA GGG CTC CCC CTG CAT CA - #C ATC AGG AGG ATG TTC         1327                                                                       Asp Gln Thr His Ser Gly Leu Pro Leu His Hi - #s Ile Arg Arg Met Phe           290                 2 - #95                 3 - #00                 3 -      #05                                                                              - - ATG GGA GAT TCA TGC CCT TAT CTA GCA GGG AA - #G CCA AAG ATG TTT        TTT     1375                                                                    Met Gly Asp Ser Cys Pro Tyr Leu Ala Gly Ly - #s Pro Lys Met Phe Phe                          310  - #               315  - #               320              - - ATT CAG AAC TAT GTG GTG TCA GAG GGC CAG CT - #G GAG GAC AGC AGC CTC         1423                                                                       Ile Gln Asn Tyr Val Val Ser Glu Gly Gln Le - #u Glu Asp Ser Ser Leu                       325      - #           330      - #           335                  - - TTG GAG GTG GAT GGG CCA GCG ATG AAG AAT GT - #G GAA TTC AAG GCT CAG         1471                                                                       Leu Glu Val Asp Gly Pro Ala Met Lys Asn Va - #l Glu Phe Lys Ala Gln                   340          - #       345          - #       350                      - - AAG CGA GGG CTG TGC ACA GTT CAC CGA GAA GC - #T GAC TTC TTC TGG AGC         1519                                                                       Lys Arg Gly Leu Cys Thr Val His Arg Glu Al - #a Asp Phe Phe Trp Ser               355              - #   360              - #   365                          - - CTG TGT ACT GCG GAC ATG TCC CTG CTG GAG CA - #G TCT CAC AGC TCA CCG         1567                                                                       Leu Cys Thr Ala Asp Met Ser Leu Leu Glu Gl - #n Ser His Ser Ser Pro           370                 3 - #75                 3 - #80                 3 -      #85                                                                              - - TCC CTG TAC CTG CAG TGC CTC TCC CAG AAA CT - #G AGA CAA GAA AGA        AAA     1615                                                                    Ser Leu Tyr Leu Gln Cys Leu Ser Gln Lys Le - #u Arg Gln Glu Arg Lys                          390  - #               395  - #               400              - - CGC CCA CTC CTG GAT CTT CAC ATT GAA CTC AA - #T GGC TAC ATG TAT GAT         1663                                                                       Arg Pro Leu Leu Asp Leu His Ile Glu Leu As - #n Gly Tyr Met Tyr Asp                       405      - #           410      - #           415                  - - TGG AAC AGC AGA GTT TCT GCC AAG GAG AAA TA - #T TAT GTT TGG CTG CAG         1711                                                                       Trp Asn Ser Arg Val Ser Ala Lys Glu Lys Ty - #r Tyr Val Trp Leu Gln                   420          - #       425          - #       430                      - - CAC ACT CTG AGA AAG AAA CTT ATC CTC TCC TA - #C ACA TAA                  - #   1750                                                                    His Thr Leu Arg Lys Lys Leu Ile Leu Ser Ty - #r Thr  *                            435              - #   440              - #   445                          - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  445 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Ser Ala Glu Val Ile His Gln Val Glu Gl - #u Ala Leu Asp Thr Asp        1               5 - #                 10 - #                 15              - - Glu Lys Glu Met Leu Leu Phe Leu Cys Arg As - #p Val Ala Ile Asp Val                   20     - #             25     - #             30                  - - Val Pro Pro Asn Val Arg Asp Leu Leu Asp Il - #e Leu Arg Glu Arg Gly               35         - #         40         - #         45                      - - Lys Leu Ser Val Gly Asp Leu Ala Glu Leu Le - #u Tyr Arg Val Arg Arg           50             - #     55             - #     60                          - - Phe Asp Leu Leu Lys Arg Ile Leu Lys Met As - #p Arg Lys Ala Val Glu       65                 - # 70                 - # 75                 - # 80       - - Thr His Leu Leu Arg Asn Pro His Leu Val Se - #r Asp Tyr Arg Val Leu                       85 - #                 90 - #                 95              - - Met Ala Glu Ile Gly Glu Asp Leu Asp Lys Se - #r Asp Val Ser Ser Leu                  100      - #           105      - #           110                  - - Ile Phe Leu Met Lys Asp Tyr Met Gly Arg Gl - #y Lys Ile Ser Lys Glu              115          - #       120          - #       125                      - - Lys Ser Phe Leu Asp Leu Val Val Glu Leu Gl - #u Lys Leu Asn Leu Val          130              - #   135              - #   140                          - - Ala Pro Asp Gln Leu Asp Leu Leu Glu Lys Cy - #s Leu Lys Asn Ile His      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Ile Asp Leu Lys Thr Lys Ile Gln Lys Ty - #r Lys Gln Ser Val        Gln                                                                                             165  - #               170  - #               175             - - Gly Ala Gly Thr Ser Tyr Arg Asn Val Leu Gl - #n Ala Ala Ile Gln Lys                  180      - #           185      - #           190                  - - Ser Leu Lys Asp Pro Ser Asn Asn Phe Arg Se - #r Ile Pro Glu Glu Arg              195          - #       200          - #       205                      - - Tyr Lys Met Lys Ser Lys Pro Leu Gly Ile Cy - #s Leu Ile Ile Asp Cys          210              - #   215              - #   220                          - - Ile Gly Asn Glu Thr Glu Leu Leu Arg Asp Th - #r Phe Thr Ser Leu Gly      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Tyr Glu Val Gln Lys Phe Leu His Leu Ser Me - #t His Gly Ile Ser        Gln                                                                                             245  - #               250  - #               255             - - Ile Leu Gly Gln Phe Ala Cys Met Pro Glu Hi - #s Arg Asp Tyr Asp Ser                  260      - #           265      - #           270                  - - Phe Val Cys Val Leu Val Ser Arg Gly Gly Se - #r Gln Ser Val Tyr Gly              275          - #       280          - #       285                      - - Val Asp Gln Thr His Ser Gly Leu Pro Leu Hi - #s His Ile Arg Arg Met          290              - #   295              - #   300                          - - Phe Met Gly Asp Ser Cys Pro Tyr Leu Ala Gl - #y Lys Pro Lys Met Phe      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Phe Ile Gln Asn Tyr Val Val Ser Glu Gly Gl - #n Leu Glu Asp Ser        Ser                                                                                             325  - #               330  - #               335             - - Leu Leu Glu Val Asp Gly Pro Ala Met Lys As - #n Val Glu Phe Lys Ala                  340      - #           345      - #           350                  - - Gln Lys Arg Gly Leu Cys Thr Val His Arg Gl - #u Ala Asp Phe Phe Trp              355          - #       360          - #       365                      - - Ser Leu Cys Thr Ala Asp Met Ser Leu Leu Gl - #u Gln Ser His Ser Ser          370              - #   375              - #   380                          - - Pro Ser Leu Tyr Leu Gln Cys Leu Ser Gln Ly - #s Leu Arg Gln Glu Arg      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Lys Arg Pro Leu Leu Asp Leu His Ile Glu Le - #u Asn Gly Tyr Met        Tyr                                                                                             405  - #               410  - #               415             - - Asp Trp Asn Ser Arg Val Ser Ala Lys Glu Ly - #s Tyr Tyr Val Trp Leu                  420      - #           425      - #           430                  - - Gln His Thr Leu Arg Lys Lys Leu Ile Leu Se - #r Tyr Thr                          435          - #       440          - #       445                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1045 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: both                                                   - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 88..1044                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GGGAAATTAA GTTTCTTGCG GAGTACGGTG GGGATTGCAG CTGCTGAGCA GG -             #GATTCTGG     60                                                                 - - AAAGCATTGC GTACCTGAGC CCCCAGC ATG GCG GGC CTA AAG - #CGG CGG GCA            111                                                                                         - #            Met Ala Gly Le - #u Lys Arg Arg Ala                            - #                  - #      450                            - - AGC CAG GTG TGG CCA GAA GAG CAT GGT GAG CA - #G GAA CAT GGG CTG TAC          159                                                                       Ser Gln Val Trp Pro Glu Glu His Gly Glu Gl - #n Glu His Gly Leu Tyr           455                 4 - #60                 4 - #65                 4 -      #70                                                                              - - AGC CTG CAC CGC ATG TTT GAC ATC GTG GGC AC - #T CAT CTG ACA CAC        AGA      207                                                                    Ser Leu His Arg Met Phe Asp Ile Val Gly Th - #r His Leu Thr His Arg                          475  - #               480  - #               485              - - GAT GTG CGC GTG CTT TCT TTC CTC TTT GTT GA - #T GTC ATT GAT GAC CAC          255                                                                       Asp Val Arg Val Leu Ser Phe Leu Phe Val As - #p Val Ile Asp Asp His                       490      - #           495      - #           500                  - - GAG CGT GGA CTC ATC CGA AAT GGA CGT GAC TT - #C TTA TTG GCA CTG GAG          303                                                                       Glu Arg Gly Leu Ile Arg Asn Gly Arg Asp Ph - #e Leu Leu Ala Leu Glu                   505          - #       510          - #       515                      - - CGC CAG GGC CGC TGT GAT GAA AGT AAC TTT CG - #C CAG GTG CTG CAG CTG          351                                                                       Arg Gln Gly Arg Cys Asp Glu Ser Asn Phe Ar - #g Gln Val Leu Gln Leu               520              - #   525              - #   530                          - - CTG CGC ATC ATC ACT CGC CAC GAC CTG CTG CC - #C TAC GTC ACC CTC AAG          399                                                                       Leu Arg Ile Ile Thr Arg His Asp Leu Leu Pr - #o Tyr Val Thr Leu Lys           535                 5 - #40                 5 - #45                 5 -      #50                                                                              - - AGG AGA CGG GCT GTG TGC CCT GAT CTT GTA GA - #C AAG TAT CTG GAG        GAG      447                                                                    Arg Arg Arg Ala Val Cys Pro Asp Leu Val As - #p Lys Tyr Leu Glu Glu                          555  - #               560  - #               565              - - ACA TCA ATT CGC TAT GTG ACC CCC AGA GCC CT - #C AGT GAT CCA GAA CCA          495                                                                       Thr Ser Ile Arg Tyr Val Thr Pro Arg Ala Le - #u Ser Asp Pro Glu Pro                       570      - #           575      - #           580                  - - AGG CCT CCC CAG CCC TCT AAA ACA GTG CCT CC - #C CAC TAT CCT GTG GTG          543                                                                       Arg Pro Pro Gln Pro Ser Lys Thr Val Pro Pr - #o His Tyr Pro Val Val                   585          - #       590          - #       595                      - - TGT TGC CCC ACT TCG GGT CCT CAG ATG TGT AG - #C AAG CGG CCA GCC CGA          591                                                                       Cys Cys Pro Thr Ser Gly Pro Gln Met Cys Se - #r Lys Arg Pro Ala Arg               600              - #   605              - #   610                          - - GGG AGA GCC ACA CTT GGG AGC CAG CGA AAA CG - #C CGG AAG TCA GTG ACA          639                                                                       Gly Arg Ala Thr Leu Gly Ser Gln Arg Lys Ar - #g Arg Lys Ser Val Thr           615                 6 - #20                 6 - #25                 6 -      #30                                                                              - - CCA GAT CCC AAG GAG AAG CAG ACA TGT GAC AT - #C AGA CTG CGG GTT        CGG      687                                                                    Pro Asp Pro Lys Glu Lys Gln Thr Cys Asp Il - #e Arg Leu Arg Val Arg                          635  - #               640  - #               645              - - GCT GAA TAC TGC CAG CAT GAG ACT GCT CTG CA - #G GGC AAT GTC TTC TCT          735                                                                       Ala Glu Tyr Cys Gln His Glu Thr Ala Leu Gl - #n Gly Asn Val Phe Ser                       650      - #           655      - #           660                  - - AAC AAG CAG GAC CCA CTT GAG CGC CAG TTT GA - #G CGC TTT AAC CAG GCC          783                                                                       Asn Lys Gln Asp Pro Leu Glu Arg Gln Phe Gl - #u Arg Phe Asn Gln Ala                   665          - #       670          - #       675                      - - AAC ACC ATC CTC AAG TCC CGG GAC CTG GGC TC - #C ATC ATC TGT GAC ATC          831                                                                       Asn Thr Ile Leu Lys Ser Arg Asp Leu Gly Se - #r Ile Ile Cys Asp Ile               680              - #   685              - #   690                          - - AAG TTC TCT GAG CTC ACC TAC CTC GAT GCA TT - #C TGG CGT GAC TAC ATC          879                                                                       Lys Phe Ser Glu Leu Thr Tyr Leu Asp Ala Ph - #e Trp Arg Asp Tyr Ile           695                 7 - #00                 7 - #05                 7 -      #10                                                                              - - AAT GGC TCT TTA TTA GAG GCA CTT AAA GGT GT - #C TTC ATC ACA GAC        TCC      927                                                                    Asn Gly Ser Leu Leu Glu Ala Leu Lys Gly Va - #l Phe Ile Thr Asp Ser                          715  - #               720  - #               725              - - CTC AAG CAA GCT GTG GGC CAT GAA GCC ATC AA - #G CTG CTG GTA AAT GTA          975                                                                       Leu Lys Gln Ala Val Gly His Glu Ala Ile Ly - #s Leu Leu Val Asn Val                       730      - #           735      - #           740                  - - GAC GAG GAG GAC TAT GAG CTG GGC CGA CAG AA - #A CTC CTG AGG AAC TTG         1023                                                                       Asp Glu Glu Asp Tyr Glu Leu Gly Arg Gln Ly - #s Leu Leu Arg Asn Leu                   745          - #       750          - #       755                      - - ATG CTG CAA GCT TTG CCC TGA A      - #                  - #                   1045                                                                     Met Leu Gln Ala Leu Pro  *                                                        760              - #   765                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  318 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Ala Gly Leu Lys Arg Arg Ala Ser Gln Va - #l Trp Pro Glu Glu His        1               5 - #                 10 - #                 15              - - Gly Glu Gln Glu His Gly Leu Tyr Ser Leu Hi - #s Arg Met Phe Asp Ile                   20     - #             25     - #             30                  - - Val Gly Thr His Leu Thr His Arg Asp Val Ar - #g Val Leu Ser Phe Leu               35         - #         40         - #         45                      - - Phe Val Asp Val Ile Asp Asp His Glu Arg Gl - #y Leu Ile Arg Asn Gly           50             - #     55             - #     60                          - - Arg Asp Phe Leu Leu Ala Leu Glu Arg Gln Gl - #y Arg Cys Asp Glu Ser       65                 - # 70                 - # 75                 - # 80       - - Asn Phe Arg Gln Val Leu Gln Leu Leu Arg Il - #e Ile Thr Arg His Asp                       85 - #                 90 - #                 95              - - Leu Leu Pro Tyr Val Thr Leu Lys Arg Arg Ar - #g Ala Val Cys Pro Asp                  100      - #           105      - #           110                  - - Leu Val Asp Lys Tyr Leu Glu Glu Thr Ser Il - #e Arg Tyr Val Thr Pro              115          - #       120          - #       125                      - - Arg Ala Leu Ser Asp Pro Glu Pro Arg Pro Pr - #o Gln Pro Ser Lys Thr          130              - #   135              - #   140                          - - Val Pro Pro His Tyr Pro Val Val Cys Cys Pr - #o Thr Ser Gly Pro Gln      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Met Cys Ser Lys Arg Pro Ala Arg Gly Arg Al - #a Thr Leu Gly Ser        Gln                                                                                             165  - #               170  - #               175             - - Arg Lys Arg Arg Lys Ser Val Thr Pro Asp Pr - #o Lys Glu Lys Gln Thr                  180      - #           185      - #           190                  - - Cys Asp Ile Arg Leu Arg Val Arg Ala Glu Ty - #r Cys Gln His Glu Thr              195          - #       200          - #       205                      - - Ala Leu Gln Gly Asn Val Phe Ser Asn Lys Gl - #n Asp Pro Leu Glu Arg          210              - #   215              - #   220                          - - Gln Phe Glu Arg Phe Asn Gln Ala Asn Thr Il - #e Leu Lys Ser Arg Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Gly Ser Ile Ile Cys Asp Ile Lys Phe Se - #r Glu Leu Thr Tyr        Leu                                                                                             245  - #               250  - #               255             - - Asp Ala Phe Trp Arg Asp Tyr Ile Asn Gly Se - #r Leu Leu Glu Ala Leu                  260      - #           265      - #           270                  - - Lys Gly Val Phe Ile Thr Asp Ser Leu Lys Gl - #n Ala Val Gly His Glu              275          - #       280          - #       285                      - - Ala Ile Lys Leu Leu Val Asn Val Asp Glu Gl - #u Asp Tyr Glu Leu Gly          290              - #   295              - #   300                          - - Arg Gln Lys Leu Leu Arg Asn Leu Met Leu Gl - #n Ala Leu Pro              305                 3 - #10                 3 - #15                            - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1200 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: both                                                   - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 32..988                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - CTGGAAAGCA CTCTATTTCT GAGCCTCTAG C ATG GCG GGC CTA - #AAG AGG CGG            52                                                                                          - #                 Met - # Ala Gly Leu Lys Arg Arg                           - #                 320 - #                 325              - - GCA AGC CAG GTG TGG CCC GAA GAG CGT GGG GA - #G CAA GAA CAT GGG CTC          100                                                                       Ala Ser Gln Val Trp Pro Glu Glu Arg Gly Gl - #u Gln Glu His Gly Leu                       330      - #           335      - #           340                  - - TAC AGC CTC CAC CGC ATG TTC GAC ATC GTG GG - #C ACC CAC CTA ACA CAC          148                                                                       Tyr Ser Leu His Arg Met Phe Asp Ile Val Gl - #y Thr His Leu Thr His                   345          - #       350          - #       355                      - - AGA GAT GTC CGA GTG CTT TCC TTC CTT TTT GT - #T GAT GTT ATT GAT GAC          196                                                                       Arg Asp Val Arg Val Leu Ser Phe Leu Phe Va - #l Asp Val Ile Asp Asp               360              - #   365              - #   370                          - - CAT GAA CGT GGA CTC ATC CGA AAT GGA CGT GA - #C TTC TTA TTG GCA CTG          244                                                                       His Glu Arg Gly Leu Ile Arg Asn Gly Arg As - #p Phe Leu Leu Ala Leu           375                 3 - #80                 3 - #85                 3 -      #90                                                                              - - GAG CGC CAG GGC CGC TGT GAC GAG AGT AAC TT - #T CGC CAG GTG CTG        CAG      292                                                                    Glu Arg Gln Gly Arg Cys Asp Glu Ser Asn Ph - #e Arg Gln Val Leu Gln                          395  - #               400  - #               405              - - CTG CTG CGC ATC ATC ACT CGC CAT GAC TTG CT - #G CCC TAC GTT ACT CTC          340                                                                       Leu Leu Arg Ile Ile Thr Arg His Asp Leu Le - #u Pro Tyr Val Thr Leu                       410      - #           415      - #           420                  - - AAG AAG AGA CGA GCT GTG TGC CCT GAT CTT GT - #A GAC AAG TAT CTG GAG          388                                                                       Lys Lys Arg Arg Ala Val Cys Pro Asp Leu Va - #l Asp Lys Tyr Leu Glu                   425          - #       430          - #       435                      - - GAA ACA TCA ATT CGC TAT GTG ACC CCC AGA GC - #C CTC AGT GAC CCA GAA          436                                                                       Glu Thr Ser Ile Arg Tyr Val Thr Pro Arg Al - #a Leu Ser Asp Pro Glu               440              - #   445              - #   450                          - - CCG AGG CCT CCC CAG CCC TCT AAA ACA GTG CC - #T CCC CAC TAT CCT GTG          484                                                                       Pro Arg Pro Pro Gln Pro Ser Lys Thr Val Pr - #o Pro His Tyr Pro Val           455                 4 - #60                 4 - #65                 4 -      #70                                                                              - - GTG TGC TGC CCC ACT TCG GGT TCT CAA ATG TG - #T AGT AAG CGG CCA        GCC      532                                                                    Val Cys Cys Pro Thr Ser Gly Ser Gln Met Cy - #s Ser Lys Arg Pro Ala                          475  - #               480  - #               485              - - CGA GGG AGA ACC ACA CTT GGG AGC CAG CGA AA - #A CGC CGG AAG TCG GTG          580                                                                       Arg Gly Arg Thr Thr Leu Gly Ser Gln Arg Ly - #s Arg Arg Lys Ser Val                       490      - #           495      - #           500                  - - ACA CCA GAC CCG AAG GAA AAG CAG ACA TGT GA - #T ATC AGG CTC CGA GTT          628                                                                       Thr Pro Asp Pro Lys Glu Lys Gln Thr Cys As - #p Ile Arg Leu Arg Val                   505          - #       510          - #       515                      - - CGG GCG GAA TAC TGC CAG CAT GAG ACG GCT CT - #G CAA GGC AAT GTC TTC          676                                                                       Arg Ala Glu Tyr Cys Gln His Glu Thr Ala Le - #u Gln Gly Asn Val Phe               520              - #   525              - #   530                          - - TCC AAT AAG CAG GAC CCA CTT GAG CGC CAG TT - #T GAG CGC TTT AAC CAG          724                                                                       Ser Asn Lys Gln Asp Pro Leu Glu Arg Gln Ph - #e Glu Arg Phe Asn Gln           535                 5 - #40                 5 - #45                 5 -      #50                                                                              - - GCC AAC ACT ATC CTC AAG TCC CGG GAC CTG GG - #C TCC ATC ATC TGT        GAC      772                                                                    Ala Asn Thr Ile Leu Lys Ser Arg Asp Leu Gl - #y Ser Ile Ile Cys Asp                          555  - #               560  - #               565              - - ATC AAG TTC TCT GAG CTC ACC TAC CTC GAC GC - #A TTC TGG CGA GAC TAC          820                                                                       Ile Lys Phe Ser Glu Leu Thr Tyr Leu Asp Al - #a Phe Trp Arg Asp Tyr                       570      - #           575      - #           580                  - - ATT AAT GGC TCA TTA TTA GAG GCA CTG AAA GG - #T GTC TTC ATC ACA GAC          868                                                                       Ile Asn Gly Ser Leu Leu Glu Ala Leu Lys Gl - #y Val Phe Ile Thr Asp                   585          - #       590          - #       595                      - - TCT CTC AAG CAA GCT GTG GGC CAT GAA GCC AT - #C AAG CTG CTG GTG AAC          916                                                                       Ser Leu Lys Gln Ala Val Gly His Glu Ala Il - #e Lys Leu Leu Val Asn               600              - #   605              - #   610                          - - GTG GAT GAG GAG GAC TAT GAG CTG GGC CGA CA - #G AAA CTC CTG AGG AAC          964                                                                       Val Asp Glu Glu Asp Tyr Glu Leu Gly Arg Gl - #n Lys Leu Leu Arg Asn           615                 6 - #20                 6 - #25                 6 -      #30                                                                              - - TTG ATG CTG CAG GCA TTA CCC TGA CCTTTCCCCT TC - #TCACCTTT CTGGGGACT    G    1018                                                                      Leu Met Leu Gln Ala Leu Pro  *                                                                635                                                            - - TTCGCTCCGT CACCTCTGGA GCTGACATAC TGTTCTGGGG TTTGTTCTCT AC -             #CCTTTCCA   1078                                                                 - - ACCAATCACA CCGCCTTTTT TTTTTTTTTT TTTTAAAAGG AAAAGACAAA GG -            #AAGGTGGA   1138                                                                 - - AGTGGTGTCC CTGCCCTCCC TGCACCCATG TGCCTGGGCT TCCCCGTTTC CT -            #GTTGCCAC   1198                                                                 - - TT                  - #                  - #                  - #                1200                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  318 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Ala Gly Leu Lys Arg Arg Ala Ser Gln Va - #l Trp Pro Glu Glu Arg        1               5 - #                 10 - #                 15              - - Gly Glu Gln Glu His Gly Leu Tyr Ser Leu Hi - #s Arg Met Phe Asp Ile                   20     - #             25     - #             30                  - - Val Gly Thr His Leu Thr His Arg Asp Val Ar - #g Val Leu Ser Phe Leu               35         - #         40         - #         45                      - - Phe Val Asp Val Ile Asp Asp His Glu Arg Gl - #y Leu Ile Arg Asn Gly           50             - #     55             - #     60                          - - Arg Asp Phe Leu Leu Ala Leu Glu Arg Gln Gl - #y Arg Cys Asp Glu Ser       65                 - # 70                 - # 75                 - # 80       - - Asn Phe Arg Gln Val Leu Gln Leu Leu Arg Il - #e Ile Thr Arg His Asp                       85 - #                 90 - #                 95              - - Leu Leu Pro Tyr Val Thr Leu Lys Lys Arg Ar - #g Ala Val Cys Pro Asp                  100      - #           105      - #           110                  - - Leu Val Asp Lys Tyr Leu Glu Glu Thr Ser Il - #e Arg Tyr Val Thr Pro              115          - #       120          - #       125                      - - Arg Ala Leu Ser Asp Pro Glu Pro Arg Pro Pr - #o Gln Pro Ser Lys Thr          130              - #   135              - #   140                          - - Val Pro Pro His Tyr Pro Val Val Cys Cys Pr - #o Thr Ser Gly Ser Gln      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Met Cys Ser Lys Arg Pro Ala Arg Gly Arg Th - #r Thr Leu Gly Ser        Gln                                                                                             165  - #               170  - #               175             - - Arg Lys Arg Arg Lys Ser Val Thr Pro Asp Pr - #o Lys Glu Lys Gln Thr                  180      - #           185      - #           190                  - - Cys Asp Ile Arg Leu Arg Val Arg Ala Glu Ty - #r Cys Gln His Glu Thr              195          - #       200          - #       205                      - - Ala Leu Gln Gly Asn Val Phe Ser Asn Lys Gl - #n Asp Pro Leu Glu Arg          210              - #   215              - #   220                          - - Gln Phe Glu Arg Phe Asn Gln Ala Asn Thr Il - #e Leu Lys Ser Arg Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Gly Ser Ile Ile Cys Asp Ile Lys Phe Se - #r Glu Leu Thr Tyr        Leu                                                                                             245  - #               250  - #               255             - - Asp Ala Phe Trp Arg Asp Tyr Ile Asn Gly Se - #r Leu Leu Glu Ala Leu                  260      - #           265      - #           270                  - - Lys Gly Val Phe Ile Thr Asp Ser Leu Lys Gl - #n Ala Val Gly His Glu              275          - #       280          - #       285                      - - Ala Ile Lys Leu Leu Val Asn Val Asp Glu Gl - #u Asp Tyr Glu Leu Gly          290              - #   295              - #   300                          - - Arg Gln Lys Leu Leu Arg Asn Leu Met Leu Gl - #n Ala Leu Pro              305                 3 - #10                 3 - #15                            - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - AGGCTGGTCT CGAACTCC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - TTCTCCAAGC AGCAATCC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GGCCTCCCAA AGTGCTGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - TTCAGGCTCC ATAATGGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Gln Ala Cys Gln Gly                                                                      5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Gln Asn Tyr Val Val                                                                      5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Gln Ala Cys Arg Gly                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Gln Ala Cys Gly Gly                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Leu Glu Val Asp Gly                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Tyr Val Ala Asp Ala Met Cys                                               - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Asp Glu Val Asp Ala Met Cys                                             __________________________________________________________________________

What is claimed is:
 1. An isolated, substantially pure protein havingthe amino acid sequence of SEQ ID NO:
 2. 2. A composition comprising theprotein of claim 1 and a pharmaceutically acceptable carrier.
 3. Anisolated, substantially pure p39 subunit of the FLAME-1 proteinconsisting of the amino acid sequence of amino acid residues 1-341 ofSEQ ID NO:
 2. 4. An isolated, substantially pure p12 subunit of FLAME-1protein consisting of the amino acid sequence of amino acid residues342-445 of SEQ ID NO: 2.